Enriched Environment Enhances the Myelin Regulatory Factor by mTOR Signaling and Protects the Myelin Membrane Against Oxidative Damage in Rats Exposed to Chronic Immobilization Stress.

Long-term consequences of stress intervene in normal signaling of the brain leading to many psychological complications. The enriched environment (EE) may potentially ameliorate the stress response in rats. However, the mechanistic understanding of the enriched environment in protecting the myelin membrane from oxidative damage after prolonged exposure to immobilization stress (IS) remains vague. In the current study, we examined the impact of EE by exposing the rats to IS (4 h/day) followed by EE treatment (2 h/day) for 28 days and the activities of ROS, lipid peroxides, and phospholipids were studied, and its influence on the myelin regulatory factor (MyRF) and enzymes linked to sphingolipid was assessed in the forebrain region of myelin membrane. The ROS and lipid peroxidation was increased, and a significant decrease in the antioxidant activities was found in the IS group. IS + EE could reduce oxidative damage and increase the levels of antioxidant activities. The individual phospholipids including sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidic acid (PA) were decreased in the IS group, while IS + EE exhibited significant increase in the phospholipid classes regardless of the exposure to IS. There was down-regulation in the mRNA levels of MyRF, CERS2, SPLTC2, UGT8, and GLTP, while IS + EE could mitigate the up-regulation in the levels of mRNA of MyRF, CERS2, SPLTC2, UGT8, and GLTP. The protein expression of MOG, PLP1, and mTOR was found to be reduced in the IS group of rats, however, IS + EE revealed significant increase in the expression of these signaling molecules. These results suggest that EE had a positive effect on chronic stress response by protecting the myelin membrane against oxidative damage and increasing the protein synthesis required for myelin membrane plasticity via activation of MyRF and mTOR signaling in the forebrain region of IS exposed rats.

Enriched environment modulates behavior, myelination and augments molecules governing the plasticity in the forebrain region of rats exposed to chronic immobilization stress.

Recently, several reports on chronic stress have shown that prolonged exposure to stress contributes to psychological and neurological complications. However, the impact of stress-induced alterations in myelination remains to be unexplored. Therefore, in the current study, the rats were subjected to immobilization stress (IS) followed by enriched environment (EE) and the behavioral, neurochemical changes pertaining to neuronal survival pathway, in addition, to the ultrastructural changes in myelin in forebrain (FB) region of rats were analyzed. Immobilization stress-exposed rats (4 h/day IS, for 28 days) exhibited increased anhedonia, anxiety, immobility, and reduced social interaction, which could be reflected in increased levels of corticosterone. In contrast, exposure to EE (4 h IS+2 h EE/day, for 28 days) was found to minimize anhedonic state, supress the depressive-like features, enhance social interaction and also reduce the levels of corticosterone. The ultrastructural changes in the FB region of the brain revealed that IS group showed enhanced g-ratio indicating decreased myelin thickness, while EE group exhibited reduced g-ratio manifesting increased myelination. Further, the study revealed that IS exposed group showed decreased levels of NGF, TrkA, PI3K, AKT, ERK, CREB, and MBP in FB regions whereas EE group could preserve normal protein and mRNA levels of these neuronal survival molecules. The results from this study suggest that EE exerts a positive impact by improving myelination in rats exposed to chronic immobilization stress.

Site specific hypermethylation of CpGs in Connexin genes 30, 26 and 43 in different grades of glioma and attenuated levels of their mRNAs.

AIM: Gliomas, the intracranial tumours are considered the deadliest malignancies. The gap junctional Connexins (Cxs) that maintain cellular homeostasis perform a unique function in glial tumour suppression. However, the differential methylation patterns of Cxs were not revealed in glioma so far. The current study attempts to categorise promoter methylation of Cx30 and Cx26 and intron methylation of Cx43 in different grades of human glioma. MATERIALS AND METHODS: About 85 glioma patients with pathologically confirmed grades and 15 control brain tissues were recruited in the study. Bisulphite-PCR-Single Stranded Conformation analysis(SSCA), Bisulphite sequencing and MeDIP-qPCR were carried out to assess methylation status and Cx mRNA levels were also analysed to evaluate the effect of methylation. RESULTS: We found that promoter CpG islands(CpGs) reside in Sp1 and Ap2 sites of Cx30 and 26 were hypermethylated in high grades (HG) of glioma rather than low grades. The input % of both was significantly increased (p < 0.03) in progressive grades. Interestingly, Cx43 could exhibit a significant increase (p < 0.05) in input % only in grade IV. While, Cx30 and 26 mRNAs were downregulated according to their methylation status in progressive fashion with grades, Cx43 was downregulated irrespective of intron methylation. CONCLUSION: Thus, we suggest that the sites and extent of methylation of Cxs (30 and 26 but not in 43) are found to be altered. In different grades of glioma can provide better appreciation of the grade of the patient and might help in strategies based on epigenetic approaches.

Implication of connexin30 on the stemness of glioma: connexin30 reverses the malignant phenotype of glioma by modulating IGF-1R, CD133 and cMyc.

Gap-junctional intercellular communication (GJIC) plays a major role in the malignant growth of glioma. Although the mechanistic aspects of GJIC have been extensively studied, the role of connexins in the regulation of the malignant behavior of glioma stem cells (GSCs) remains unclear. In our previous studies, we have shown that connexin30 can interfere with the insulin-like growth factor 1 receptor (IGF-1R), which is known for self-renewal and pluripotency. Following our earlier in vitro observation, in this work, we aimed to study the consequence of this influence of Cx30 on IGF-1R by evaluating the marker of GSCs, CD133 and oncoprotein, cMyc. We strengthened our basis by examining human glioma samples of different grades as well as rat C6 xenografts (Cx30-transfected and -non-transfected C6 cells) along with the sphere formation assays in vitro. Investigation of stemness-related CD133 and cMyc in human samples and rat xenografts exhibited a reciprocal relationship between Cx30 and IGF-1R in the low and high grades (HG) of glioma. Cx30 was completely abolished in HG; levels of IGF-1R, CD133 and cMyc expression were positively correlated with HG. Cx30 transfection could attenuate the malignant burden of glioma in rat xenografts. Cx30 transfection also altered the tumor sphere formation of C6 glioma cells in vitro, an important property of GSCs, and there was a significant reduction of CD133 and cMyc expression by Cx30 both in vitro and in vivo. These factors indicate that dysfunction of Cx30 plays a crucial role in the prevention of the stemness of glioma, and the exploitation of this feature will help in the management of glioma.

Nardostachys jatamansi Targets BDNF-TrkB to Alleviate Ketamine-Induced Schizophrenia-Like Symptoms in Rats.

OBJECTIVE: Schizophrenia, a common neurological disorder appearing in the late teens or early adulthood, is characterized by disorganized thinking, behaviour, and perception of emotions. Aberrant N-methyl-D-aspartate (NMDA) receptor-mediated synaptic plasticity is a major pathological event here due to dysfunction of dopamine and glutamate transmission at NMDA receptors. De-regulated brain-derived neurotrophic factor (BDNF), i.e., its signalling through the tropomyosin receptor kinase B (TrkB) receptor, is a major feature of schizophrenia. With recent global awareness of traditional plant medicines in reducing side effects, the aim of our study was to evaluate the efficacy of the ethanolic root extract of a herb belonging to the Valerianacea family, Nardostachys jatamansi, against ketamine-induced schizophrenia-like model in rats. METHODS: The effect of the N. jatamansi drug (oral dosage of 500 mg/kg body weight for 14 days) in ketamine-administered male Wistar albino rats (30 mg/kg body weight for 5 days) on modulating behaviour and the level of neurotransmitters like dopamine and glutamate was studied in whole-brain homogenates, and its influence on BDNF and TrkB levels in 2 relevant brain regions, the hippocampus and prefrontal cortex, was assessed. RESULTS: We observed that N. jatamansi treatment exhibited encouraging results in the modulation of ketamine-induced schizophrenia-like behaviours, principally the positive symptoms. Our drug both significantly upregulated the glutamate level and downregulated the dopamine level in whole-brain homogenates and retained the normal levels of BDNF (in the hippocampus but not in the prefrontal cortex) and TrkB (in both hippocampus and prefrontal cortex) induced by ketamine in rats. CONCLUSION: These findings suggest a neuroprotective effect of the ethanolic root extract of N. jatamansi against ketamine-induced schizophrenia-like symptoms in rats; possibly, regarding its effect on TrkB signalling. Further research is warranted in the treatment of schizophrenic symptoms.

Targeted Metabolic Profiling in Determining the Metabolic Heterogeneity in Human Biopsies of Different Grades of Glioma.

Gliomas are intricate tumors with numerous metabolic and genetic abnormalities contributing to their aggressive phenotypes and poor prognoses. The study aims at identifying the key molecular metabolic as well as gene expressional variations that could be used to differentiate between different grades of glioma to obtain deeper insights the about metabolic status of glioma that may serve as good candidates of diagnosis in future. In the present study, the metabolomic profiling along with clinical and expressional analyses of glioma biopsies (n = 52; patients comprising both of benign and malignant lesions) was analyzed. The biopsies were subjected to gene/protein expressional analysis using RT-PCR and western blotting and also were subjected to metabolite analyses. The results of the gene/protein expressional analysis exhibited elevated levels of carnitine palmitoyltransferase, monoglyceride lipase, human phosphofructokinase, and isocitrate dehydrogenase in higher grades of glioma when compared to those of control. Our study suggested that the metabolites and gene/protein expressional levels were found to be discriminative among the grades of glioma. The study is deemed as a provider of deeper insights that are essential for differential therapeutic approaches that specifically target the dysregulated metabolome to the benefit of patients.

Plausible role of naringenin against cerebrally implanted C6 glioma cells in rats.

Gliomas encompass a significant percentage of intrinsic neoplasms of the central nervous system in both adults and children. The constitutive activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B is the hallmark of glioma. The up-regulated protein kinase B could influence the expression of cyclooxygenase-2, an indicator of aggressive glioma. The present study was embarked to demonstrate the effect of naringenin (50 mg/kg bw for 30 days administrated orally) on PI3K, protein kinase B, and cyclooxygenase-2 in cerebrally implanted rat C6 glioma model. After the experimental period of 30 days, the animals were sacrificed and excised brain tissues were subjected to study the expressions of PI3K, protein kinase B, and cyclooxygenase-2 by reverse transcriptase polymerase chain reaction followed Western blot analysis. The activity of COX-2 (production of prostaglandin-E(2)) was also determined by high pressure liquid chromatography. The results showed that the naringenin could down-regulate the expressions of PI3K and protein kinase B along with activity and expression of cyclooxygenase-2 in C6 glioma cells implanted rat brain. In conclusion, it can be argued that the reduced expressions of phosphatidylinositol 3-kinase and protein kinase B in naringenin-treated glioma-induced rat brain might be involved in the down-regulation of cyclooxygenase-2 expression and activity. Thus, fine-tuned investigation of which will be helpful for targeted drug discovery against glioma.

Enriched Environment Minimizes Anxiety/Depressive-Like Behavior in Rats Exposed to Immobilization Stress and Augments Hippocampal Neurogenesis (In Vitro).

Chronic exposure to stress disturbs the homeostasis of the brain, thus, deleteriously affecting the neurological circuits. In literature, there are investigations about the stress-related alterations in behavioral response and adult neurogenesis; however, an effective combating strategy to evade stress is still at stake. Hence, the present study is designed to investigate the effect of an enriched environment in alleviating the anxiety/depressive-like behavioral response and enhancing the adult neurogenesis in the hippocampal region of rats exposed to chronic immobilization stress. The rats were exposed to chronic immobilization stress (IS) for 4 h/day followed by the enriched environment (EE) for 2 h/day for 28 days, and finally, the hippocampal region was dissected out after the behavioral analyses. IS group showed increased behavioral despair to tail suspension test, decrement in the activity for light/dark box test, and less grooming activity towards splash test. In contrast, IS + EE rats exhibited a decrease in the activity of tail suspension test and an increase in the behavioral response to light/dark box test and splash test. The in vitro assessment of primary cultures of neurospheres from the IS group resulted in decreased levels of proliferation in the cell number and metabolic activity of both MTT assay and lactate levels. IS + EE group revealed an increase in the growth curve of neurospheres and higher metabolic activities of MTT and lactate. The IS cultures had reduced neurite length, while the neurite outgrowths were increased in IS + EE group. The IS group showed significant reduction in the protein and mRNA levels of nestin, GFAP, CD11b, MOG, and synaptophysin, whereas the IS + EE cultures exhibited significant increase in the levels of these stem cell markers. Our data highlight the positive impact of EE against stress-related behavioral changes in rats exposed to chronic immobilization stress perhaps by interfering with the differentiation of neurospheres and neurogenesis.

Naringenin promote apoptosis in cerebrally implanted C6 glioma cells.

Naringenin (NGEN), a naturally occurring citrus flavonone, has shown cytotoxicity in various human cancer cell lines as well as inhibitory effects on tumor growth. It has been also shown to access the brain and there is an increasing interest in its therapeutic applications. The up-regulated expression of Cx43 leads to the suppression of tumorigenicity with promoted apoptotic events. In this study, we investigated the in vivo effect of NGEN in fostering apoptosis in cerebrally implanted C6 glioma cells rat model. We analysed the expression of Cx43, caspase-3, caspase-9, Cyt C, Bcl-2 and Bax in vivo by immunoblot analysis and the ultra structure of brain cells by transmission electron microscopy. Supplementation of NGEN to experimental animals modulated Bcl-2/Bax ratio and up-regulation of caspase-3 and 9. NGEN was also found to up-regulate the expression of Cx43. These findings provide evidence that NGEN's apoptotic effect, modulation of Bcl-2/Bax ratio leads to release of Cyt C from mitochondria, thereby activation of caspase-3 and caspase-9 is mediated by enhanced expression of Cx43. These observations were well supported by the transmission electron microscopic results which showed the characteristic apoptotic features. In conclusion, our findings demonstrate that NGEN promotes apoptosis in rat C6 glioma model.

Naringenin Sensitizes Resistant C6 Glioma Cells with a Repressive Impact on the Migrating Ability.

BACKGROUND: Glioma, the most common form of a malignant brain tumour is characterised by a poor prognosis, which is attributable to its resistance against current therapeutic approaches. Temozolomide (TMZ), a DNA alkylating agent, is the first-line drug for glioma treatment. Long-term treatment using TMZ was reported to culminate in the development of resistance with overexpression of multidrug resistance 1 gene coded protein P-glycoprotein, which in turn releases the drugs from the tumour cells. PURPOSE: Thus, to circumvent such resistance issues, the current study attempted to explore the effect of naringenin (a flavanone) with proven antiglial tumour potential, in mitigating the features of TMZ resistance. METHODS: Colony-forming assay, invasion assay and scratch wound assay were performed among the groups, namely tumour control (C6), vehicle control (V), naringenin (NGEN)-treated, drug-resistant tumour cells (C6R), and drug resistance cells added with NGEN (C6R+NGEN), to examine the impact of NGEN on migration and invasion. The effect of NGEN on filopodia length and density during cell migration was also studied in addition to the matrix metalloproteinases (MMP-2 and MMP-9) and p-ERK levels. RESULTS AND CONCLUSION: NGEN and C6R+NGEN groups had shown significant reduction (P < .01) in length and density of filopodia, colony formation, invasion and wound healing. Further, NGEN could also modify the assessed protein levels (P < .001), which were involved in migration and invasion in sensitive and resistant cells. Our study had provided the first evidence on NGEN-induced enhanced sensitivity against TMZ resistance with profound influence as an antimigratory and anti-invasive agent.

Dopamine, a key factor of mitochondrial damage and neuronal toxicity on rotenone exposure and also parkinsonic motor dysfunction-Impact of asiaticoside with a probable vesicular involvement.

Persuasive evidence propose that the toxicity of dopamine in parkinsonism and the loss of dopaminergic neurons are the earliest events during the pathogenesis of Parkinson's disease (PD). In our earlier study, Asiaticoside (AS), a triterpenoid saponin isolated from Centella asiatica was shown to exert a neuroprotective effect against hemiparkinsonism, purportedly due to phosphoinositides (PI)-assisted cytodynamics and synaptic function. Here, we evaluate AS in the modulation of dopamine (DA), mitochondrial integrity and neurite variations in vitro and motor dysfunctions in vivo. PC12 cells challenged with rotenone-(ROT) (0.1 µM/mL) were exposed to AS and l-DOPA (10 mM and 20 µM/mL respectively). The protein expressions of Bax and Bcl-2 that regulate cell death were assessed following neurite length assays. Rats were distributed into 6 groups (6 rats/group): Sham, Vehicle controls, ROT-infused (6 µg/µl/kg), AS- treated (50 mg/kg/day), Drug control, and ROT + L-DOPA-treated (6 mg/kg/day) groups. At the end of the experimental period, the rats were sacrificed after performing motor behavioral analysis, and the striatum was dissected out. The contents of synaptic vesicular and cytosolic DA were analyzed. Further, the levels of striatal PI were also measured. ROT had caused significant reduction in the neurite outgrowth in the exposed PC12 cells while the tested concentrations of AS and l-DOPA can exert their protective effect on the stunted neurite growth. The levels of Bax, Bcl-2, and cytochrome c which were significantly disturbed by ROT, could also be affected by AS thereby suggesting its effect on neurons. AS treatment caused an improved motor performance, vesicular and cytosolic DA, and striatal PI. These pre-clinical findings force us to speculate that AS could be a potential drug candidate in combating ROT-induced variations that are possibly precipitated by varied vesicular trafficking of DA.

The novel phytocomponent asiaticoside-D isolated from Centella asiatica exhibits monoamine oxidase-B inhibiting potential in the rotenone degenerated cerebral ganglions of Lumbricus terrestris.

BACKGROUND: Centella asiatica (CA) is one of the most valuable herbal medicines widely being used for the treatment of various neurological ailments that are challenging for health-care providers and also is deemed to be safe and effective. PURPOSE: Monoamines (MAs) are neurotransmitters and neuromodulators that play a significant role in the neural communication, regulation of motor and cognitive functions in the brain. Neurodegeneration is associated with elevated levels of MAO-B that can lead to damaging reactive oxygen species (ROS) in the brain. The current study, evaluated the effects of asiaticoside-D (AD) from neuroprotective CA, on the levels and activities of monoamine oxidase A and B (MAO-A and B), in addition to the behavioral analysis. METHODS: Qualitative and quantitative analyses of various solvent extracts of CA were performed. The extracts were screened for antioxidant potential using 1,1-diphenyl-2-picryl hydroxyl (DPPH), 2,2'-azinobis-3-ethylbenzothiozoline-6-sulfonic acid (ABTS), hydrogen peroxide (H2O2) radical, nitric oxide (NO) radical inhibition, lipid peroxidation (LPO) and ferric reducing antioxidant power (FRAP) assays. The purification of AD was done by column, thin layer and high-performance liquid chromatographies followed by structural elucidation using IR, HR-MS, 1H and 13C NMR spectra. Docking studies were performed to assess the impact of AD on MAO-A and B.In vivo, Lumbricus terrestris were exposed to 0.4 ppm rotenone (ROT) of medium for 7 days and were subjected to co-treatment along with 15 ppm of AD from CA. At the end of experiment period, the neuronal behavior of worms was assessed. Cerebral ganglions (CGs) were removed and the m-RNA levels of MAO-A and B were analyzed by Semi Q-PCR and their activities were also analyzed. RESULTS: The ethanolic extracts exhibited higher antiradical activity against DPPH, ABTS, H2O2, LPO, FRAP, NO and vitamin C with EC50 value of 20.2, 20.9, 20.4, 22.0, 24.9, 28.1, 25.5 and 22.0 µg/ml respectively. Structural analysis by IR, HR-MS, 1H and 13C NMR spectrum have shown the structure of the isolated compound as (2α, 3ß)-2,3-dihydroxyurs-12-en-28-oicacid-O-α-L-rhamnopyranosyl-(1→4)-O-ß-d-glucopyranosyl (1→6)-ß-copyranosyl ester and was represented as AD. In silico interaction of AD with MAO-A and B residues Lys312 at distances of 1.84 Šand 2.44 Šrespectively was found to exhibit high binding energy of -9.4 and 7.4 kcal. The neuronal behavior using L. terrestris showed significant improvement against (p < 0.001) ROT impaired behavior (group II) on AD supplementation (p < 0.05). Further, the m-RNA levels and activities of MAO-A and B which were significantly altered (p < 0.001) by ROT could be effectively maintained on AD supplementation. CONCLUSION: AD was found to exert its negative impact on the levels and activities of MAO-A and B in CGs of rotenone- induced changes in L. terrestris, the property which is considered to be crucial against ROT induced neurodegenerative pathology like -Parkinsonism.

Counter effects of Asiaticosids-D through putative neurotransmission on rotenone induced cerebral ganglionic injury in Lumbricus terrestris.

Asiaticoside-D (AD) was shown to efficacy of ganglionic degenerated Lumbricus terrestris as a pioneering observation in our earlier research. Though, extract molecular mechanisms of AD for degenerative diseases (DDs) remains largely unknown. We investigated the neuroprotective effects of AD against ROT in cerebral ganglions (CGs) of degenerative L. terrestris. Worms were exposed to 0.4 ppm ROT for 7 days were subjected to co- treatment with 15 ppm of AD. After, CGs was removed. The levels oxidant, non-antioxidant, antioxidant status, ganglioside, ceramide and ceramide glycanase (CGase) were estimated. The m-RNA levels of dopamine transporter (DAT), octopamine transporter (OAT), innexins-9 (inx-9), ionotropic glutamate receptor 3 (iGlu3), heat shock proteins (hsp70), XPRLamide neuropeptide precursor, tyramine beta-hydroxylase (tbh-1) and ß- adrenergic receptor kinase-2 (ß-ARK2-3) by semi-qRT- PCR. The expression pattern of tyramine beta hydroxylase (TBH), glutamate receptor (iGluR), serotonin transporter (SERT), dopamine transporters (DAT), nerve growth factors (NGF), cytochrome C oxidase (COC), NADH dehydogenase subunit-1 (ND-1), neurotrophin receptor p75 (p75NTR), neuronal nitric oxiside synthase (nNOs) interleukin 1- beta (IL1-ß) and tumor necrosis factor alpha (TNF-α) by western blotting. Glutaminergic, serotogenic and dopaminergic toxicity variations were also performed. The levels of oxidant, non-antioxidant, antioxidant status, lipids, proteins and m-RNAs were significantly altered (p < 0.001) on ROT-induced (group II) and their levels were significantly changes (p < 0.05) by ROT+AD in CGs. The sensitive study plan concluded the neuroprotective effects of AD against ROT induced degeneration in worms and suggest that the AD deserves future studies for its use as an effective alternative medicine that could minimize the morbidity of ganglionic degenerative diseases patients.

Homology modeling identified for purported drug targets to the neuroprotective effects of levodopa and asiaticoside-D in degenerated cerebral ganglions of Lumbricus terrestris.

CONTEXT: Homology modeling plays role in determining the therapeutic targets dreadful for condition such as neurodegenerative diseases (NDD), which pose challenge in achieving the effective managements. The structures of the serotonin transporter (SERT), aquaporin (AQP), and tropomyosin receptor kinase (TrkA) which are implicated in NDD pathology are still unknown for Lumbricus terrestris, but the three-dimensional (3D) structure of the human counterpart for modeling. AIM: This study aims to generate and evaluate the 3D structure of TrkA, SERT, and AQP proteins and their interaction with the ligands, namely Asiaticoside-D (AD) and levodopa (L-DOPA) the anti-NDD agents. SUBJECTS AND METHODS: Homology modeling for SERT, AQP, and TrkA proteins of Lumbricus terrestris using SWISS-MODEL Server and the modeled structure was validated using Rampage Server. Wet-lab analysis of their correspondent m-RNA levels was also done to validate the in silico data. RESULTS: It was found that TrkA had moderately high homology (67%) to human while SERT and AQP could exhibit 58% and 42%, respectively. The reliability of the model was assessed by Ramachandran plot analysis. Interactions of AD with the SERT, AQP-4, and TrkA showed the binding energies as -9.93, 8.88, and -7.58 of Kcal/mol, respectively, while for L-DOPA did show -3.93, -5.13, and -6.0 Kcal/mol, respectively. The levels of SERT, TrkA, and AQP-4 were significantly reduced (P < 0.001) on ROT induced when compared to those of control worms. On ROT + AD supplementation group (III), m-RNA levels were significantly increased (P < 0.05) when compared to those of ROT induced worms (group II). CONCLUSION: Our pioneering docking data propose the possible of target which is proved useful for therapeutic investigations against the unconquered better of NDD.

In vitro assessment of curcumin against murine neuroblastoma cells.

OBJECTIVE: Neuroblastoma (NB) is a well-known malignant disease in infants, which comprises 10% of childhood malignancies. Despite recent advances in understanding the neuro-oncology, NB still accounts for more death in childhood than any other cancer. Research in childhood tumors should not only be focused on the malignant signatures of cancer cells but also novel drug prototypes using phytochemicals. The present study was aimed to determine the role of curcumin against murine neuroblastoma cell line (N2a). METHODS: The in vitro assessment of curcumin against was made in N2a cell line in a dose-dependent manner (group I (control) and group II - IX (10 microM-80 microM). The efficacy of the drug was evaluated by estimating the levels of protein bound carbohydrates, glycoprotein, genomic DNA, total RNA levels, and inhibition of MMP-9 were studied. The gap junctional communication in the cells was also assessed. RESULTS: The levels of protein bound carbohydrates, DNA, RNA levels, glycoprotein were found to be altered on drug supplementation in NB cells. Inhibition of MMP-9 in curcumin-supplemented N2a cells was revealed by zymographic analysis. Assessment of Lucifer yellow dye uptake in curcumin-supplemented N2a cells showed the up-regulation of GJIC. CONCLUSIONS: These observations suggest that the curcumin, the active principle of curcuma longa, could be developed into an effective chemo preventive and chemotherapeutic agent. This selected concentration range needs further studies at molecular level, for conforming its role and its action against uncontrolled proliferation of NB.

Attenuated levels of phospholipids in the striatum of rats infused with rotenone causing hemiparkinsonism as detected by simple dye-lipid complex.

Parkinson's disease (PD), a progressive neurodegeneration, is characterized by loss of dopaminergic neurons in the substantia nigra (SN) and loss of motor co-ordination. Impaired metabolism of major lipids such as phospholipids which play regulatory roles in cellular functions and signaling has been implicated in the pathology of PD. We aim to investigate the striatal phospholipids (PLs) in hemiparkinsonism infused by rotenone in rats. As there are no cost-effective modes of PL, we have utilized dye-lipid complex technique for the first time in PD models for screening and also for semi-quantifying (individually) the levels of the deregulated PL in brain samples. Rats were divided into 2 groups: i. control and ii. ROT-infused which received intracranial injection of Rotenone (6 µg/µl; flow rate 0.2 µl/min). At the end of experimental period of 14 days, the striatum was dissected out for the analyses of PLs. Dye-based detection of PL and two-dimensional thin-layer chromatographic analyses of PL were performed. Detection of dye-PL complex was possible for phosphatidyl choline (PC), phosphatidyl inositol (PI), and spingomyelin (SM) (but not for phosphatidyl ethanolamine-PE) using dyes viz victoria blue B, toluidine blue and ammonium ferrothiocyanate, respectively. Two-dimensional analyses of phospholipids confirmed the dye-PL complex and depicted significant reduction (p < 0.05) on semi-quantitative assessment, in the striatum of control and hemiparkinsonic rats. We suggest a low level of PLs esp of PI in striatum of rats using a simple dye-detection that was validated by HR-LCMS. The finding implies that a critical role is being played by these PLs (PC, PI and SM) mainly PI (p < 0.001), in rotenone infused hemiparkinsonism, thus deserving wider but simpler investigations to detect and identify their role in parkinsonism.

Rotenone causing dysfunctional mitochondria and lysosomes in cerebral ganglions of Lumbricus terrestris degenerate giant fibers and neuromuscular junctions.

Rotenone is well-documented to cause neurodegenerative condition such as Parkinson's, in the exposed systems. However, its detrimental effect on particular sites of neuronal pathway is still under investigation. We aimed at elucidating the impact of rotenone on cerebral ganglions (CG) of Lumbricus terrestris which control movement and behaviour of the worms. Worms were exposed to 0-0.4 ppm/mL of rotenone. Mitochondrial and lysosomal integrities were found to be affected beyond 0.2 ppm/mL of rotenone. Activities of cholinergic enzymes and the expression of tyrosine hydroxylase showed an impaired neuronal transmission in CGs at the dose of 0.2 ppm/mL of rotenone. Histopathological and immunoflourescent analyses showed neuronal apoptosis, reduced nucleic acid content and inhibited of neurosecretion at 0.3 ppm/mL. Electron microscopy showed that the neurons and neuromuscular junctions were affected at 0.2 ppm/mL. Dose-dependent changes were also observed in the motor function such as burrowing behaviours and locomotion. Conduction velocity (CV) and locomotion assessment showed that the CV of lateral giant fiber (LGF) was reduced while that of MGF remains unaffected at 0.2 ppm, the dose at which the burrowing behaviour was also not affected. LGF, cholinergic enzymes and tyrosine hydroxylase are primarily targeted by rotenone affecting locomotion at 0.2 ppm/mL while MGF, neuropile and the burrowing behaviour were affected at 0.3 ppm/mL. We demonstrate, in addition to dose-dependent effects, that the bioaccumulation factors range 0.28-0.32 ppm/µg of rotenone cause degenerative impact on giant fibers affecting neuronal behaviors/locomotion of worms. We also propose worms for studying mechanisms of neuronal pathology caused by chemicals prevailing in earth's atmosphere.

Connexin 30 downregulates Insulin-like growth factor receptor-1, abolishes Erk and potentiates effects of an IGF-R inhibitor in a glioma cell line.

Connexins (Cx) play a crucial role in cell communication though regulation of cell growth and proliferation. In recent decades, both suppressive and enhancing roles of gap junction proteins in malignancy have been proposed, though mechanisms remain unclear. We intend to evaluate the impact of Cx30 on dysregulated growth of glioma owing to an aberrant expression of Insulin-like growth factor-1 receptor (IGF-1R). The study also examined whether Cx30 expression influenced sensitivity of glioma cells to Picropodophyllin (PPP), the potent inhibitor of IGF-1R. C6 cells transfected with full length Cx30 resulted in complete abolition of colony-forming efficiency. Interestingly, PPP-supplemented cells behaved differently with and without exogenous Cx as confirmed by wound closure assay. The expressions of phosphorylated and unphosphorylated IGF-1R along with its key signaling enzymes, pAkt/pErk, were also varied significantly in transfected and non-transfected C6 cells. pIGF-1R and IGF-1R were significantly reduced on Cx30 transfection when compared with that of non-transfected cells. pErk expression was abolished in transfected C6 with no significant difference in the expression of pAkt. The potency of PPP against C6 was more pronounced in the presence of Cx30. We demonstrate that Cx30 has the potential to alter the IGF-1R mediated pathway thereby influencing the growth, proliferation and migration of glioma cells which could further enhance the effect of therapeutic intervention. Though it could not be corroborated that the observations made are due to Cx30-mediated channel-dependent and/or independent impact, we stress the impact of significance of Cx30 on IGF-1R in glioma and also in therapeutic aspects.