Hypothalamic GABA receptor functional regulation and liver cell proliferation

GABAergic alterations in hypothalamus during compensatory hyperplasia after partial hepatectomy (PH), lead nitrate (LN) induced direct hyperplasia and N-nitrosodiethylamine (NDEA) induced neoplasia in liver were investigated. Serum GABA levels were increased in all 3 experimental groups compared with the control. GABA content decreased in hypothalamus of PH and NDEA treated rats, while it increased in LN treated rats. GABAA receptor number and affinity in hypothalamic membrane preparations of rats showed a significant decrease in PH and NDEA treated rats, while in LN treated rats the affinity increased without any change in the receptor number. The GABAB receptor number increased in PH and NDEA treated rats, while it decreased in LN treated rats. The affinity of the receptor also increased in NDEA treated rats. Plasma NE levels showed significant increase in PH and NDEA rats compared with the control while it decreased in LN treated rats. The results of the present study suggests that liver cell proliferation is influencing the hypothalamic GABAergic neurotransmission and these changes regulate the hepatic proliferation through the sympathetic stimulation.

Effect of Pyridoxine on Growth, Metabolism and Cellular Activity of Macrobrachium rosenbergii

Effect of pyridoxine on growth, metabolism and cellular activity of freshwater prawn Macrobrachiuni rosenbergii was studied. Postlarvae (PL-10) of M. rosenbergii were fed with clam meat containing various concentrations of pyridoxine. After 30 days RNA and DNA of the abdominal tissues were estimated. Length, weight and RNA to DNA ratio increased significantly with increasing concentrations of pyridoxine. The effect of pyridoxine on the metabolic enzyme, malate dehydrogenase, was also studied. Vmax showed a significant decrease and the (Km) showed a significant increase in experimental groups compared to control.

Gaba Receptor Gene Expression during Rat Liver Cell Proliferation and Its Function in Hepatocyte Cultures

The present thesis is an attempt to understand the role of GABA, GABAA and GABAB receptors in the regulation of liver cell proliferation using in vivo and in vitro models. The work also focuses on the brain GABAergic changes associated with normal and neoplastic cell growth in liver and to delineate its regulatory function. The investigation of mechanisms involving mitogenic models without cell necrosis may contribute our knowledge about both on cell growth, carcinogenesis, liver pathology and treatment. Objectives of the present study are, to induce controlled liver cell proliferation by partial hepatectomy and lead nitrate administration and uncontrolled cell proliferation by N-nitrosodiethylamine treatment in male Wistar rats, the changes in the content of GABA, GABAA,GABAB in various rat brain regions. To study the GABAA and GABAB receptor changes in brain stem, hypothalamus, cerebellum and cerebral cortex during the active cortex during the period of active DNA synthesis in liver of different experimental groups. The changes in GABAA and GABAB receptor function of the brain stem, hypothalamus and cerebellum play an important role sympathetic regulation of cell proliferation and neoplastic growth in liver. The decrease in GABA content in brain stem, hypothalamus and cerebellum during regeneration and neoplasia in liver. The time course of brain GABAergic changes was closely correlated with that of heptic DNA synthesis. The functional significance of these changes was further explored by studying the changes in GABAA and GABAB receptors in brain.

Adrenergic and Glutamergic Receptor Gene Expression and Their Functional regulation in the Cerebral Cortex of Hypoxia Induced Neonatal Rats:Role of Oxygen,Epinephrine and Glucose Supplementation

In the present work, the role of oxygen, epinephrine and glucose supplementation in regulating neurotransmitter contents, adrenergic and glutamate receptor binding parameters in the cerebral cortex of experimental groups of neonatal rats were investigated. The study of neurotransmitters and their receptors in the cerebral cortex and the EEG pattern in the brain regions of neonatal rats were taken as index for brain damage due to hypoxia, oxygen and epinephrine. Real-Time PCR work was done to confirm the binding parameters. Second messenger, cyclic Adenosine Monophosphate (cAMP) was assayed to find the functional correlation of the receptors. Behavioural studies were carried out to confirm the biochemical and molecular studies. The efficient and timely supplementation of glucose plays a crucial role in correcting the molecular changes due to hypoxia, oxygen and epinephrine. The addictive neuronal damage effect due to oxygen and epinephrine treatment is another important observation. The corrective measures from the molecular study brought to practice will lead to maintain healthy intellectual capacity during the later developmental stages, which has immense clinical significance in neonatal care.

Serotonin receptor subtypes functional regulation and oxidative stress mediated apoptosis in 6-hydroxydopamine lesioned Parkinsonian rats

Parkinson’s disease is a chronic progressive neurodegenerative disorder characterized by the selective loss of dopaminergic neurons in the SNpc resulting in severe motor impairments. Serotonergic system plays an important regulatory role in the pathophysiology of PD in rats, the evaluation of which provides valuable insight on the underlying mechanisms of motor, cognitive and memory deficits in PD. We observed a decrease in 5-HT content in the brain regions of 6-OHDA infused rat compared to control. The decreased 5-HT content resulted in a decrease of total 5-HT, 5-HT2A receptors and 5-HTT function and an increase of 5-HT2C receptor function. 5-HT receptor subtypes - 5-HT2A and 5-HT2C receptors have differential regulatory role on the modulation of DA neurotransmission in different brain regions during PD. Our observation of impaired serotonergic neurotransmission in SNpc, corpus striatum, cerebral cortex, hippocampus, cerebellum and brain stem demonstrate that although PD primarily results from neurodegeneration in the SNpc, the associated neurochemical changes in other areas of the brain significantly contributes to the different motor and non motor symptoms of PD. The antioxidant enzymes – SOD, CAT and GPx showed significant down regulation which indicates increased oxidative damage resulting in neurodegeneration. We also observed an increase in the level of lipid peroxidation. Reduced expression of anti-apoptotic Akt and enhanced expression of NF-B resulting from oxidative stress caused an activation of caspase-8 thus leading the cells to neurodegeneration by apoptosis. BMC administration in combination with 5-HT and GABA to PD rats showed reversal of the impaired serotonergic neurotransmission and oxidative stress mediated apoptosis. The transplanted BMC expressed NeuN confirming that 5-HT and GABA induced the differentiation and proliferation of BMC to neurons in the SNpc along with an increase in DA content and an enhanced expression of TH. Neurotrophic factors – BDNF and GDNF rendered neuroprotective effects accompanied by improvement in behavioural deficits indicating a significant reversal of altered dopaminergic and serotonergic neurotransmission in PD. The restorative and neuroprotective effects of BMC in combination with 5-HT and GABA are of immense therapeutic significance in the clinical management of PD.

Investigation on key molecular targets responsible for antidiabetic properties of Symplocos cochinchinensis (Lour.) S. Moore

Diabetes mellitus is a heterogeneous metabolic disorder characterized by hyperglycemia with disturbances in carbohydrate, protein and lipid metabolism resulting from defects in insulin secretion, insulin action or both. Currently there are 387 million people with diabetes worldwide and is expected to affect 592 million people by 2035. Insulin resistance in peripheral tissues and pancreatic beta cell dysfunction are the major challenges in the pathophysiology of diabetes. Diabetic secondary complications (like liver cirrhosis, retinopathy, microvascular and macrovascular complications) arise from persistent hyperglycemia and dyslipidemia can be disabling or even life threatening. Current medications are effective for control and management of hyperglycemia but undesirable effects, inefficiency against secondary complications and high cost are still serious issues in the present prognosis of this disorder. Hence the search for more effective and safer therapeutic agents of natural origin has been found to be highly demanding and attract attention in the present drug discovery research. The data available from Ayurveda on various medicinal plants for treatment of diabetes can efficiently yield potential new lead as antidiabetic agents. For wider acceptability and popularity of herbal remedies available in Ayurveda scientific validation by the elucidation of mechanism of action is very much essential. Modern biological techniques are available now to elucidate the biochemical basis of the effectiveness of these medicinal plants. Keeping this idea the research programme under this thesis has been planned to evaluate the molecular mechanism responsible for the antidiabetic property of Symplocos cochinchinensis, the main ingredient of Nishakathakadi Kashayam, a wellknown Ayurvedic antidiabetic preparation. A general introduction of diabetes, its pathophysiology, secondary complications and current treatment options, innovative solutions based on phytomedicine etc has been described in Chapter 1. The effect of Symplocos cochinchinensis (SC), on various in vitro biochemical targets relevant to diabetes is depicted in Chapter 2 including the preparation of plant extract. Since diabetes is a multifactorial disease, ethanolic extract of the bark of SC (SCE) and its fractions (hexane, dichloromethane, ethyl acetate and 90 % ethanol) were evaluated by in vitro methods against multiple targets such as control of postprandial hyperglycemia, insulin resistance, oxidative stress, pancreatic beta cell proliferation, inhibition of protein glycation, protein tyrosine phosphatase-1B (PTP-1B) and dipeptidyl peptidase-IV (DPPxxi IV). Among the extracts, SCE exhibited comparatively better activity like alpha glucosidase inhibition, insulin dependent glucose uptake (3 fold increase) in L6 myotubes, pancreatic beta cell regeneration in RIN-m5F and reduced triglyceride accumulation in 3T3-L1 cells, protection from hyperglycemia induced generation of reactive oxygen species in HepG2 cells with moderate antiglycation and PTP-1B inhibition. Chemical characterization by HPLC revealed the superiority of SCE over other extracts due to presence of bioactives (beta-sitosterol, phloretin 2’glucoside, oleanolic acid) in addition to minerals like magnesium, calcium, potassium, sodium, zinc and manganese. So SCE has been subjected to oral sucrose tolerance test (OGTT) to evaluate its antihyperglycemic property in mild diabetic and diabetic animal models. SCE showed significant antihyperglycemic activity in in vivo diabetic models. Chapter 3 highlights the beneficial effects of hydroethanol extract of Symplocos cochinchinensis (SCE) against hyperglycemia associated secondary complications in streptozotocin (60 mg/kg body weight) induced diabetic rat model. Proper sanction had been obtained for all the animal experiments from CSIR-CDRI institutional animal ethics committee. The experimental groups consist of normal control (NC), N + SCE 500 mg/kg bwd, diabetic control (DC), D + metformin 100 mg/kg bwd, D + SCE 250 and D + SCE 500. SCEs and metformin were administered daily for 21 days and sacrificed on day 22. Oral glucose tolerance test, plasma insulin, % HbA1c, urea, creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, total protein etc. were analysed. Aldose reductase (AR) activity in the eye lens was also checked. On day 21, DC rats showed significantly abnormal glucose response, HOMA-IR, % HbA1c, decreased activity of antioxidant enzymes and GSH, elevated AR activity, hepatic and renal oxidative stress markers compared to NC. DC rats also exhibited increased level of plasma urea and creatinine. Treatment with SCE protected from the deleterious alterations of biochemical parameters in a dose dependent manner including histopathological alterations in pancreas. SCE 500 exhibited significant glucose lowering effect and decreased HOMA-IR, % HbA1c, lens AR activity, and hepatic, renal oxidative stress and function markers compared to DC group. Considerable amount of liver and muscle glycogen was replenished by SCE treatment in diabetic animals. Although metformin showed better effect, the activity of SCE was very much comparable with this drug. xxii The possible molecular mechanism behind the protective property of S. cochinchinensis against the insulin resistance in peripheral tissue as well as dyslipidemia in in vivo high fructose saturated fat diet model is described in Chapter 4. Initially animal were fed a high fructose saturated fat (HFS) diet for a period of 8 weeks to develop insulin resistance and dyslipidemia. The normal diet control (ND), ND + SCE 500 mg/kg bwd, high fructose saturated fat diet control (HFS), HFS + metformin 100 mg/kg bwd, HFS + SCE 250 and HFS + SCE 500 were the experimental groups. SCEs and metformin were administered daily for the next 3 weeks and sacrificed at the end of 11th week. At the end of week 11, HFS rats showed significantly abnormal glucose and insulin tolerance, HOMA-IR, % HbA1c, adiponectin, lipid profile, liver glycolytic and gluconeogenic enzyme activities, liver and muscle triglyceride accumulation compared to ND. HFS rats also exhibited increased level of plasma inflammatory cytokines, upregulated mRNA level of gluconeogenic and lipogenic genes in liver. HFS exhibited the increased expression of GLUT-2 in liver and decreased expression of GLUT-4 in muscle and adipose. SCE treatment also preserved the architecture of pancreas, liver, and kidney tissues. Treatment with SCE reversed the alterations of biochemical parameters, improved insulin sensitivity by modifying gene expression in liver, muscle and adipose tissues. Overall results suggest that SC mediates the antidiabetic activity mainly via alpha glucosidase inhibition, improved insulin sensitivity, with antiglycation and antioxidant activities.