Anurag Tripathi

Curcumin-Loaded Nanoparticles Potently Induce Adult Neurogenesis and Reverse Cognitive Deficits in Alzheimer’s Disease Model via Canonical Wnt/β-Catenin Pathway

Neurogenesis, a process of generation of new neurons, is reported to be reduced in several neurodegenerative disorders including Alzheimers disease (AD). Induction of neurogenesis by targeting endogenous neural stem cells (NSC) could be a promising therapeutic approach to such diseases by influencing the brain self-regenerative capacity. Curcumin, a neuroprotective agent, has poor brain bioavailability. Herein, we report that curcumin-encapsulated PLGA nanoparticles (Cur-PLGA-NPs) potently induce NSC proliferation and neuronal differentiation in vitro and in the hippocampus and subventricular zone of adult rats, as compared to uncoated bulk curcumin. Cur-PLGA-NPs induce neurogenesis by internalization into the hippocampal NSC. Cur-PLGA-NPs significantly increase expression of genes involved in cell proliferation (reelin, nestin, and Pax6) and neuronal differentiation (neurogenin, neuroD1, neuregulin, neuroligin, and Stat3). Curcumin nanoparticles increase neuronal differentiation by activating the Wnt/β-catenin pathway, involved in regulation of neurogenesis. These nanoparticles caused enhanced nuclear translocation of β-catenin, decreased GSK-3β levels, and increased promoter activity of the TCF/LEF and cyclin-D1. Pharmacological and siRNA-mediated genetic inhibition of the Wnt pathway blocked neurogenesis-stimulating effects of curcumin. These nanoparticles reverse learning and memory impairments in an amyloid beta induced rat model of AD-like phenotypes, by inducing neurogenesis. In silico molecular docking studies suggest that curcumin interacts with Wif-1, Dkk, and GSK-3β. These results suggest that curcumin nanoparticles induce adult neurogenesis through activation of the canonical Wnt/β-catenin pathway and may offer a therapeutic approach to treating neurodegenerative diseases such as AD, by enhancing a brain self-repair mechanism.

Interactive threats of nanoparticles to the biological system.

The use of nanoscale materials is growing exponentially, but concerns rise about the human hazards cannot be ignored. Nanotechnology has penetrated deep into our lives in diversified areas as engineering, information technology and diagnostics. Nonetheless owing to their peculiar properties these new materials also present new health risks upon interacting with biological systems. This is a typical case of technology preceding toxicity and therefore, various toxicological aspects for an array of nanomaterials are just beginning to be assessed. Several deleterious effects are being noticed, particularly in vitro situations as well as in mammalian system. Nanoparticles toxicity is compellingly related to oxidative stress, alteration of calcium homeostasis, gene expression, pro-inflammatory responses and cellular signalling events. It is therefore critical to understand the nature and origin of the toxicity imposed by nanomaterials. Keeping all these points in mind, the present review provides updated information on the various aspects such as sources of production, effect of different physical properties, interaction with biological system and mechanisms of engineered nanoparticles induced toxicities.

Activation of Autophagic Flux against Xenoestrogen Bisphenol-A-induced Hippocampal Neurodegeneration via AMP kinase (AMPK)/Mammalian Target of Rapamycin (mTOR) Pathways

Background: The effects of xenoestrogen bisphenol-A on autophagy, and association with oxidative stress and apoptosis are still elusive. Results: Transient activation of autophagy protects against bisphenol-A-induced neurodegeneration via AMPK activation and mTOR down-regulation. Conclusion: Autophagy induction against bisphenol-A is an early cells tolerance response. Significance: Autophagy provides an imperative biological marker for evaluation of neurotoxicity by xenoestrogen. The human health hazards related to persisting use of bisphenol-A (BPA) are well documented. BPA-induced neurotoxicity occurs with the generation of oxidative stress, neurodegeneration, and cognitive dysfunctions. However, the cellular and molecular mechanism(s) of the effects of BPA on autophagy and association with oxidative stress and apoptosis are still elusive. We observed that BPA exposure during the early postnatal period enhanced the expression and the levels of autophagy genes/proteins. BPA treatment in the presence of bafilomycin A1 increased the levels of LC3-II and SQSTM1 and also potentiated GFP-LC3 puncta index in GFP-LC3-transfected hippocampal neural stem cell-derived neurons. BPA-induced generation of reactive oxygen species and apoptosis were mitigated by a pharmacological activator of autophagy (rapamycin). Pharmacological (wortmannin and bafilomycin A1) and genetic (beclin siRNA) inhibition of autophagy aggravated BPA neurotoxicity. Activation of autophagy against BPA resulted in intracellular energy sensor AMP kinase (AMPK) activation, increased phosphorylation of raptor and acetyl-CoA carboxylase, and decreased phosphorylation of ULK1 (Ser-757), and silencing of AMPK exacerbated BPA neurotoxicity. Conversely, BPA exposure down-regulated the mammalian target of rapamycin (mTOR) pathway by phosphorylation of raptor as a transient cells compensatory mechanism to preserve cellular energy pool. Moreover, silencing of mTOR enhanced autophagy, which further alleviated BPA-induced reactive oxygen species generation and apoptosis. BPA-mediated neurotoxicity also resulted in mitochondrial loss, bioenergetic deficits, and increased PARKIN mitochondrial translocation, suggesting enhanced mitophagy. These results suggest implication of autophagy against BPA-mediated neurodegeneration through involvement of AMPK and mTOR pathways. Hence, autophagy, which arbitrates cell survival and demise during stress conditions, requires further assessment to be established as a biomarker of xenoestrogen exposure.

In vitro studies on immunotoxic potential of Orange II in splenocytes.

Orange II, an azo dye, is not permitted in food preparations, but high levels of the dye have been detected in different food commodities. Though there are reports on the toxicity of Orange II but knowledge based on the immunomodulatory properties of Orange II is scanty. The present investigation was undertaken to study the in vitro immunotoxic potential of Orange II in splenocytes. Splenocytes were isolated, cultured and subjected to immunophenotypic analysis, mixed lymphocyte reaction (MLR) assay or stimulated with lipopolysaccharide (LPS) or concanavalin A (Con A) for 72 h. The supernatant was collected for cytokine assays. Orange II showed cytotoxic effects at 100-1000μg/ml concentrations and 50μg/ml was determined as the highest non-cytotoxic dose. Orange II at the non-cytotoxic dose (50μg/ml) significantly altered the relative distribution of T and B-cells, MLR response and the mitogen induced proliferative response of T-cells and B-cells. Consistent with the hypo-responsiveness of the T and B-lymphocytes, Orange II induced a concomitant decline in the secretion of cytokines IL-2, IL-4, IL-6, IFN-γ, TNF-α and IL-17. On the contrary, there was an increase in the production of IL-10, an anti-inflammatory regulatory cytokine, which may be one of the causative factor for immunosuppressive property of Orange II. These results suggest that non-cytotoxic dose of Orange II may have immunomodulatory effects.

Bisphenol-A Mediated Inhibition of Hippocampal Neurogenesis Attenuated by Curcumin via Canonical Wnt Pathway

Bisphenol A (BPA) is an environmental xenoestrogenic endocrine disruptor, utilized for production of consumer products, and exerts adverse effects on the developing nervous system. Recently, we found that BPA impairs the finely tuned dynamic processes of neurogenesis (generation of new neurons) in the hippocampus of the developing rat brain. Curcumin is a natural polyphenolic compound, which provides neuroprotection against various environmental neurotoxicants and in the cellular and animal models of neurodegenerative disorders. Here, we have assessed the neuroprotective efficacy of curcumin against BPA-mediated reduced neurogenesis and the underlying cellular and molecular mechanism(s). Both in vitro and in vivo studies showed that curcumin protects against BPA-induced hippocampal neurotoxicity. Curcumin protects against BPA-mediated reduced neural stem cells (NSC) proliferation and neuronal differentiation and enhanced neurodegeneration. Curcumin also enhances the expression/levels of neurogenic and the Wnt pathway genes/proteins, which were reduced due to BPA exposure in the hippocampus. Curcumin-mediated neuroprotection against BPA-induced neurotoxicity involved activation of the Wnt/β-catenin signaling pathway, which wasxa0confirmed by the use of Wnt specific activators (LiCl and GSK-3β siRNA) and inhibitor (Dkk-1). BPA-mediated increased β-catenin phosphorylation, decreased GSK-3β levels, and β-catenin nuclear translocation were significantly reversed by curcumin, leading to enhanced neurogenesis. Curcumin-induced protective effects on neurogenesis were blocked by Dkk-1 in NSC culture treated with BPA. Curcumin-mediated enhanced neurogenesis was correlated well with improved learning and memory in BPA-treated rats. Overall, our results conclude that curcumin provides neuroprotection against BPA-mediated impaired neurogenesis via activation of the Wnt/β-catenin signaling pathway.

Mechanism of rhein-induced apoptosis in rat primary hepatocytes: beneficial effect of cyclosporine A.

Past observational and toxicity studies have established an association between the deaths of children and consumption of Cassia occidentalis (CO) seeds. We recently reported chemical evidence of this association following the identification of toxic anthraquinones (AQs), viz. aloe-emodin, chrysophanol, emodin, physcion, and rhein, in CO seeds (Panigrahi, G. K. et al. (2015), Chem. Res. Toxicol. DOI: 10.1021/acs.chemrestox.5b00056 ). Of these five AQs, earlier studies have shown rhein to be the most cytotoxic AQ in hepatocytes. Therefore, the present study was designed to investigate the effect of rhein on rat primary hepatocytes. Results indicated that rhein (50 μM) causes apoptosis in rat primary hepatocytes by generating reactive oxygen species (ROS), increasing intracellular Ca(2+), decreasing the mitochondrial membrane potential, and depleting intracellular glutathione content. At the molecular level, rhein-induced DNA damage results in overexpression of γ-H2AX protein (2.5-fold), thereby causing enhancement of p53 (4.5-fold) and p21 (3.6-fold), leading to intrinsic pathway-mediated apoptosis involving Bax, bcl2, cytochrome c, caspases 3 and 9, and poly-ADP ribose polymerase. Further, it was observed that rhein-induced ROS generation is also involved in the modulation of signaling molecules like MAPK kinases, including ERK1/2, p38, and JNK, and mitochondrial energetics proteins, including complexes II-V, p-AMPK, and Sirt-1. It was shown that 100 nM cyclosporine A was the most effective among the different protective agents at preventing apoptosis in hepatocytes by interfering in various metabolic pathways which were found to be altered by rhein.

Bt Brinjal in India: A long way to go

Brinjal occupies the major proportion amongst all vegetable crops in India and is vulnerable to many diseases caused by insect-pests, fungus, bacteria and virus. Brinjal production is extensively affected by the insect brinjal fruit and shoot borer. Use of conventional chemical pesticides not only damage environment including the biotic and abiotic components but, also affect human health. Bt Brinjal was developed to combat brinjal fruit and shoot borer that has an advantage minimizing use of chemical pesticides. Extensive biosafety investigations, nutritional studies, substantial equivalence studies, relative toxicity and allergenicity assessment using animal models like Sprague Dawley rats, Brown Norway rats, rabbit, fish, chicken, goats, etc. revealed no significant differences between genetically modified brinjal and its native counterpart. Bt brinjal could effectively control the target pest and was found to be safe for environment and human health. In spite of all the scientific studies, release of Bt Brinjal has been put under moratorium. Indian government has constituted an expert committee to address this issue. In this review we have tried to explore the facts related to Bt Brinjal including its production, use of Bt toxin, use of chemical pesticides in controlling the FSB in native brinjal, along with perspective of public opinion and government initiatives. Key words: Bt Brinjal, agriculture, insecticides, GM foods, agrobacterium, transgenic crops

A novel function of TLR4 in mediating the immunomodulatory effect of Benzanthrone, an environmental pollutant

Our prior studies have reported that Benzanthrone (BA) manifests inflammatory responses in the spleen of Balb/c mice. The present investigation was carried out to study the impact of BA on macrophages, which are the primary scavenger cells in the body that act as a connecting link between innate and adaptive immunity. Parenteral administration of BA (daily for one week) to mice resulted in enhanced levels of nitric oxide (NO) and overexpression of inflammatory markers (COX-2, MMP-9 and PGE-2) in macrophages; however the level of MHC class-I and MHC class-II receptors were down regulated. Further, the potential membrane receptor targets (TLRs) of BA and its interaction with TLRs was investigated using computational methods. Professional phagocytes play pivotal roles in sensing bacteria through pathogen-associated molecular patterns (PAMPs) by various pathogen recognition receptors (PRRs), including Toll-like receptors (TLRs). Several studies have implicated these TLRs in the amplification of the inflammatory responses, however the fundamental role played by TLRs in mediating the inflammation associated with xenobiotics is still obscure and not understood. From the in silico analysis, it was evident that BA showed the highest binding affinity with TLR4 as compared to other TLRs. The western blotting studies confirmed that BA exposure indeed upregulated the expression of TLR 4, 5 and 9. Moreover, the downstream signaling cascade proteins of TLRs such as myeloid differentiation primary response protein-88 (MyD88), IL-1 receptor associated kinase (IRAK-1), and TNFR-associated factor (TRAF-6) were found to be enhanced in the BA treated groups. It was also observed that BA treatment increased the expression of ICAM-1, p-Lyn, p-Syk, p-PI3-K, IP3, PLC-γ, cAMP and Ca+2 influx, which are known to play a critical role in TLR mediated inflammation. Earlier we found that toxic effects of BA in spleen were mediated by oxidative stress which was partially neutralized by NAC exposure. Hereby, we report that NAC treatment in conjunction with BA attenuated the expression of BA induced TLR4, as well as the inflammatory markers such as COX2 and p-NFkB in macrophages. These findings demonstrated the critical role of TLRs in the regulation of the BA-induced inflammation.

Immunomodulatory potential of Rhein, an anthraquinone moiety of Cassia occidentalis seeds

Rhein, the most toxic anthraquinone moiety in Cassia occidentalis seeds, has been associated with hepatomyoencephalopathy (HME) in children. Structural and functional alterations in the lymphoid organs have been reported both in HME patients and experimental animals indicating a possibility of the dysfunction of immune system following exposure to CO seeds or its toxic anthraquinones (Panigrahi et al., 2014a). In the present study the mechanism of immune response of Rhein in splenocytes has been investigated by measuring functional assays of lymphocyte, cell surface receptor expression and analysis of cytokine levels. Results indicate that Rhein at a maximum dose of 10 μM is non cytotoxic up to 72 h in splenocytes. In addition to its potential to decrease the allogenic response of T-cells, Rhein significantly suppresses the proliferation of the concavalin A (Con A) and lipopolysaccharide (LPS) stimulated splenocytes. Lymphocyte receptor expression analysis revealed that Rhein exposure significantly down regulate the expression of CD3e, CD4, CD8, CD28, CD69 molecules in T-cells. The expression of CD19, CD28, CD40 in B-cells were also found to be significantly decreased following Rhein exposure. In accordance with the functional responses, Rhein treatment significantly lowered the expression of IL2 and IL6 cytokines in Con A stimulated splenocytes, and IL6, IL10, IFNγ and TNFα in LPS stimulated splenocytes. Over all, the study suggests the immunomodulatory activity of Rhein and that it would be useful in understanding the immune response of CO seeds in human subjects.

Interaction of anthraquinones of Cassia occidentalis seeds with DNA and Glutathione

Graphical abstract