Abhishek Kumar Singh

Chitosan/TiO2 composite membrane improves proliferation and survival of L929 fibroblast cells: Application in wound dressing and skin regeneration

The microbial infection and growth of fibroblasts are the critical factors for the effective wound healing. The natural polymer-based dressing membranes may mimic extracellular matrix to support the survival, proliferation and differentiation of fibroblasts. The present study deals with the preparation of chitosan/titanium dioxide (CS/TiO2) composite membranes with different degree of TiO2 incorporation, and their characterization in terms of morphology, ultrastructure, thermal behavior and mechanical properties with SEM, FTIR, XRD and tensile strength analyses. The data demonstrated the formation of strong O-Ti-O bonding between TiO2 and CS resulting in superior porosity, mechanical strength, crystallinity and flexibility of the composite membranes. Further, the cyto-compatibility, proliferation, oxidative stress, cell cycle and apoptosis analyses of fibroblast L929 cells demonstrated the enhanced proliferation and survival, and decreased oxidative stress and apoptosis in L929 cells grown on CS/TiO2 membrane incorporated with 025% TiO2. Next, we measured the significant up-regulation in the expression of fibroblast-markers in L929 cells cultured on CS/TiO2 (0.25%) membrane. Furthermore, the CS/TiO2 composite membranes exhibited a superior antibacterial activity against Staphylococcus aureus. Taken together, the data confirmed that CS/TiO2 (0.25%) membrane improved the growth, survival and functional integrity of fibroblasts, and exerted antibacterial activity which may be utilized as potential dressing materials.

Neuroprotection Through Rapamycin-Induced Activation of Autophagy and PI3K/Akt1/mTOR/CREB Signaling Against Amyloid-β-Induced Oxidative Stress, Synaptic/Neurotransmission Dysfunction, and Neurodegeneration in Adult Rats

Autophagy is a catabolic process involved in the continuous removal of toxic protein aggregates and cellular organelles to maintain the homeostasis and functional integrity of cells. The mechanistic understanding of autophagy mediated neuroprotection during the development of neurodegenerative disorders remains elusive. Here, we investigated the potential role of rapamycin-induced activation of autophagy and PI3K/Akt1/mTOR/CREB pathway(s) in the neuroprotection of amyloid-beta (Aβ1-42)-insulted hippocampal neurons in rat model of Alzheimer’s disease (AD) like phenotypes. A single intra-hippocampal injection of Aβ1-42 impaired redox balance and markedly induced synaptic dysfunction, neurotransmission dysfunction, and cognitive deficit, and suppressed pro-survival signaling in the adult rats. Rapamycin administration caused a significant reduction of mTOR complex 1 phosphorylation at Ser2481 and a significant increase in levels of autophagy markers such as microtubule-associated protein-1 light chain-3 (LC3), beclin-1, sequestosome-1/p62, unc-51-like kinase 1 (ULK1). In addition, rapamycin induced the activation of autophagy that further activated p-PI3K, p-Akt1 (Ser473), and p-CREB (Ser183) expression in Aβ1-42-treated rats. The activated autophagy markedly reversed Aβ1-42-induced impaired redox homeostasis by decreasing the levels of prooxidants—ROS generation, intracellular Ca2+ flux and LPO, and increasing the levels of antioxidants—SOD, catalase, and GSH. The activated autophagy also provided significant neuroprotection against Aβ1-42-induced synaptic dysfunction by increasing the expression of synapsin-I, synaptophysin, and PSD95; and neurotransmission dysfunction by increasing the levels of CHRM2, DAD2 receptor, NMDA receptor, and AMPA receptor; and ultimately improved cognitive ability in rats. Wortmannin administration significantly reduced the expression of autophagy markers, p-PI3K, p-Akt1, and p-CREB, as well as the autophagy mediated neuroprotective effect. Our study demonstrate that autophagy can be an integrated part of pro-survival (PI3K/Akt1/mTOR/CREB) signaling and autophagic activation restores the oxidative defense mechanism(s), neurodegenerative damages, and maintains the integrity of synapse and neurotransmission in rat model of AD.

Autophagy Activation Alleviates Amyloid-β-Induced Oxidative Stress, Apoptosis and Neurotoxicity in Human Neuroblastoma SH-SY5Y Cells

Autophagy is an evolutionary conserved catabolic process that ensures continuous removal of damaged cell organelles and long-lived protein aggregates to maintain cellular homeostasis. Although autophagy has been implicated in amyloid-β (Aβ) production and deposition, its role in pathogenesis of Alzheimer’s disease remains elusive. Thus, the present study was undertaken to assess the cytoprotective and neuroprotective potential of autophagy on Aβ-induced oxidative stress, apoptosis and neurotoxicity in human neuroblastoma SH-SY5Y cells. The treatment of Aβ1-42 impaired the cell growth and redox balance, and induced apoptosis and neurotoxicity in SH-SY5Y cells. Next, the treatment of rapamycin (RAP) significantly elevated the expression of autophagy markers such as microtubule-associated protein-1 light chain-3 (LC3), sequestosome-1/p62, Beclin-1, and unc-51-like kinase-1 (ULK1) in SH-SY5Y cells. RAP-induced activation of autophagy notably alleviated the Aβ1-42-induced impairment of redox balance by decreasing the levels of pro-oxidants such as reactive oxygen species, lipid peroxidation and Ca2+ influx, and concurrently increasing the levels of antioxidant enzymes such as superoxide dismutase and catalase. The RAP-induced autophagy also ameliorated Aβ1-42-induced loss of mitochondrial membrane potential and apoptosis. Additionally, the activated autophagy provided significant neuroprotection against Aβ1-42-induced neurotoxicity by elevating the expression of neuronal markers such as synapsin-I, PSD95, NCAM, and CREB. However, 3-methyladenine treatment significantly exacerbated the neurotoxic effects of Aβ1-42. Taken together, our study demonstrated that the activation of autophagy provided possible neuroprotection against Aβ-induced cytotoxicity, oxidative stress, apoptosis, and neurotoxicity in SH-SY5Y neuronal cells.

Progress in the Development and Applicability of Potential Medicinal Plant Extract‐Conjugated Polymeric Constructs for Wound Healing and Tissue Regeneration

Wound, burn and tissue related diseases are the most common and devastating forms of trauma worldwide and thousands are still dying each year when they are left untreated. The traditional treatments for wound infection using medicinal plant extracts in hydrogels and ointment formulations have several disadvantages, delicate shape and dry up quickly upon exposure to air. Indeed, there is need for the development of an alternative form of dressing material for wound healing applications. Because the medicinal plant products are economical, researchers have adopted a novel approach of complexing the active components of plants with various groups of polymers to develop biodegradable fabrications. Moreover, fabricated constructs are very extremely useful as scaffold in tissue regeneration with known successes in wound healing/ dressing applications that the fabricated substitutes mimic the extracellular matrix of tissue. In this review, we give an extensive overview on scientifically evaluated bioactive molecules of medicinal plants as well as plant extract blended polymeric constructs for the possible treatment of various skin injuries. In addition, the technological challenges and future trends for recent developments of the treatments of wound infections are extensively summarized. Copyright

Whey protein concentrate supplementation protects rat brain against aging-induced oxidative stress and neurodegeneration

Whey protein concentrate (WPC) is a rich source of sulfur-containing amino acids and is consumed as a functional food, incorporating a wide range of nutritional attributes. The purpose of this study is to evaluate the neuroprotective effect of WPC on rat brain during aging. Young (4 months) and old (24 months) male Wistar rats were supplemented with WPC (300 mg/kg body weight) for 28 days. Biomarkers of oxidative stress and antioxidant capacity in terms of ferric reducing antioxidant potential (FRAP), lipid hydroperoxide (LHP), total thiol (T-SH), protein carbonyl (PC), reactive oxygen species (ROS), nitric oxide (NO), and acetylcholinesterase (AChE) activity were measured in brain of control and experimental (WPC supplemented) groups. In addition, gene expression and histopathological studies were also performed. The results indicate that WPC augmented the level of FRAP, T-SH, and AChE in old rats as compared with the old control. Furthermore, WPC-treated groups exhibited significant reduction in LHP, PC, ROS, and NO levels in aged rats. WPC supplementation also downregulated the expression of inflammatory markers (tumor necrosis factor alpha, interleukin (IL)-1β, IL-6), and upregulated the expression of marker genes associated with autophagy (Atg3, Beclin-1, LC3B) and neurodegeneration (neuron specific enolase, Synapsin-I, MBP-2). The findings suggested WPC to be a potential functional nutritional food supplement that prevents the progression of age-related oxidative damage in Wistar rats.

Understanding the molecular mechanism of improved proliferation and osteogenic potential of human mesenchymal stem cells grown on a polyelectrolyte complex derived from non-mulberry silk fibroin and chitosan

The development of engineered bone tissue, as a promising alternative to conventional bone grafts, has so far not proven successful and still remains challenging. Thus, attempts have been made in the present study to synthesize polyelectrolyte complex (PEC) scaffolds by blending chitosan (CS) to silk fibroin (SF) derived from the non-mulberry silkworm (Antheraea pernyi) at three different pH values (5.0, 6.0, and 7.0), and to characterize them in terms of morphology, ultrastructure and mechanical properties with scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy, x-ray diffraction and tensile strength analyses. The prepared PEC scaffolds showed a mean pore size of 130 μm, as revealed by SEM analysis, and a comparatively higher compressive strength. The findings of in vitro cytocompatibility, in vivo biocompatibility and osteogenic marker (genes/proteins) analysis suggest that the PECs blended at pH 7.0 showed greater stability and enhanced growth and an osteogenic differentiation capability of human mesenchymal stem cells (MSCs). To aid our understanding of protein-polyion binding mechanisms, we employed a molecular docking and simulation study of SF macrodomains and CS oligomer using Schrödinger 14 and GROMACS (Groningen Machine for Chemical Simulations) software. The study involved analytical techniques for macromolecular solution characterization and theoretical simulations based on molecular dynamics. The computational studies confirmed the presence of an integral RGD sequence that played a vital role in superior cell-attachment, proliferation and osteogenic differentiation of MSCs grown on the developed SF-CS PEC scaffolds.The development of engineered bone tissue, as a promising alternative to the conventional bone grafts, is not rewarding yet and remained challenging. Thus, attempts have been made in the present study to synthesize polyelectrolyte complex (PEC) scaffolds by blending of chitosan (CS) to silk fibroin (SF) derived from non-mulberry silkworm (Antheraea pernyi) at three different pH (5.0, 6.0, and 7.0), and characterize in terms of morphology, ultrastructure and mechanical properties with SEM, FTIR, XRD and tensile strength analyses. The prepared PEC scaffolds showed mean pore size of 130 µm as revealed by SEM analysis and comparatively higher compressive strength. The findings of in vitro cytocompatibility, in vivo biocompatibility and osteogenic markers (genes/proteins) analysis suggested that the PECs blended at pH 7.0 showed greater stability and enhanced growth and osteogenic differentiation capability of human mesenchymal stem cells (MSCs). To aid our understanding of protein-polyion binding mechanisms, we employed a molecular docking and simulation study of SF macrodomains and CS oligomer using Schrödinger 14 and GROMACS (GROningen Machine for Chemical Simulations) software. The study involved analytical techniques for macromolecular solution characterization and theoretical simulations based on molecular dynamics. The computational studies confirmed the presence of integral RGD sequence that played a vital role in superior cell-attachment, proliferation and osteogenic differentiation of MSCs grown on the developed SF-CS PEC scaffolds.

Rapamycin mitigates erythrocyte membrane transport functions and oxidative stress during aging in rats

Abstract Erythrocyte membrane is a suitable model to study various metabolic and physiological functions as it undergoes variety of biochemical changes during aging. An age-dependent modulatory effect of rapamycin on erythrocyte membrane functions is completely unknown. Therefore, the present study was undertaken to investigate the effect of rapamycin on age-dependent impaired activities of transporters/exchangers, altered levels of redox biomarkers, viz. protein carbonyl (PC), lipid hydroperoxides (LHs), total thiol (–SH), sialic acid (SA) and intracellular calcium ion [Ca2+]i, and osmotic fragility of erythrocyte membrane. A significant reduction in membrane-bound activities of Na+/K+-ATPase (NKA) and Ca2+-ATPase (PMCA), and levels of –SH and SA was observed along with a simultaneous induction in Na+/H+ exchanger (NHE) activity and levels of [Ca2+]i, PC, LH and osmotic fragility in old-aged rats. Rapamycin was found to be a promising age-delaying drug that significantly reversed the aging-induced impaired activities of membrane-bound ATPases and altered levels of redox biomarkers.

Fisetin as a caloric restriction mimetic protects rat brain against aging induced oxidative stress, apoptosis and neurodegeneration

Aim: In the present study, attempts have been made to evaluate the potential role of fisetin, a caloric restriction mimetic (CRM), for neuroprotection in D‐galactose (D‐gal) induced accelerated and natural aging models of rat. Main methods: Fisetin was supplemented (15 mg/kg b.w., orally) to young, D‐gal induced aged (D‐gal 500 mg/kg b.w subcutaneously) and naturally aged rats for 6 weeks. Standard protocols were employed to measure pro‐oxidants, antioxidants and mitochondrial membrane potential in brain tissues. Gene expression analysis with reverse transcriptase‐polymerase chain reaction (RT‐PCR) was performed to assess the expression of autophagy, neuronal, aging as well as inflammatory marker genes. We have also evaluated apoptotic cell death and synaptosomal membrane‐bound ion transporter activities in brain tissues. Key findings: Our data demonstrated that fisetin significantly decreased the level of pro‐oxidants and increased the level of antioxidants. Furthermore, fisetin also ameliorated mitochondrial membrane depolarization, apoptotic cell death and impairments in the activities of synaptosomal membrane‐bound ion transporters in aging rat brain. RT‐PCR data revealed that fisetin up‐regulated the expression of autophagy genes (Atg‐3 and Beclin‐1), sirtuin‐1 and neuronal markers (NSE and Ngb), and down‐regulated the expression of inflammatory (IL‐1&bgr; and TNF‐&agr;) and Sirt‐2 genes respectively in aging brain. Significance: The present study suggests that fisetin supplementation may provide neuroprotection against aging‐induced oxidative stress, apoptotic cell death, neuro‐inflammation, and neurodegeneration in rat brain.

Multinutrient Approach to Slow Down Brain Aging and Related Neurodegenerative Disorders

Abstract Old age is the greatest risk factor for most of the neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and stroke, all marked by global deficits in cognitive abilities and impaired neuronal function. The pharmacological interventions for age-related neurodegenerative disorders are limited and not yet rewarding, and also associated with significant risk for adverse effects. Multinutrients including micronutrients, macronutrients (protein and amino acids, fats, carbohydrates, vitamins, and omega-3 polyunsaturated fatty acids), and other nutrients such as secondary plant metabolites may prolong the onset of dementia and slow down brain aging. Recent findings suggest that dietary supplementation with caloric restriction mimetics and vegetable or fruit extracts exerts their beneficial effects by decreasing the age-dependent vulnerability toward oxidative stress and neurodegeneration. The present chapter provides an extensive overview on the potential of multinutrients to prevent neurodegenerative events and improve cognitive function during aging.

N-acetyl L-cysteine attenuates oxidative damage and neurodegeneration in rat brain during aging

N-acetyl-l-cysteine (NAC) is a precursor of cysteine, which is known to increase the level of glutathione (GSH) in the brain. Several neurodegenerative changes linked to oxidative stress take place in the aging brain. This study aimed to assess the neuroprotective effect of NAC supplementation on age-dependent neurodegeneration in the rat brain. Young (4 months) and old (24 months) Wistar rats (n = 6 rats/group) were supplemented with NAC (100 mg/kg b.w. orally) for 14 days. Enzymatic and nonenzymatic antioxidants such as superoxide dismutase and catalase, and GSH and total thiol respectively, prooxidants such as protein carbonyl, advanced oxidation protein products, reactive oxygen species, and malondialdehyde were assessed in the brain homogenates. Furthermore, nitric oxide level, acetylcholinesterase activity, and Na+/K+-ATPase activity were measured and gene expression studies were also performed. The results indicated that NAC augmented the level of enzymatic and nonenzymatic antioxidants with a significant reduction in prooxidant levels in old rats. NAC supplementation also downregulated the expression of inflammatory markers (TNF-α, IL-1β, IL-6) and upregulated the expression of marker genes associated with aging (sirtuin-1) and neurodegeneration (neuron-specific enolase, neuroglobin, synapsin-I, myelin basic protein 2) in old rats. The present findings support a neuroprotective role of NAC which has therapeutic implication in controlling age-related neurological disorders.