Development of Parkinson's Disease Model and Exploring Role of C-Phycocyanin Against Rotenone Induces Parkinson's Disease Model in Mice

Abstract

Background: Parkinson's Disease is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons in the basal ganglia, leading to motor symptoms such as tremors, bradykinesia, and postural instability. The disease is associated with the degeneration of substantia nigra pars compacta dopaminergic neurons, iron accumulation in the brain, presence of α-synuclein with Lewy bodies, and absence of neuromelanin. Diabetes mellitus and insulin resistance, both prevalent age-related illnesses, increase the risk and impact the progression of Parkinson's disease. While FDA-approved medications provide symptomatic relief such as Levodopa, MAO-B inhibitors, COMT inhibitors etc. Researchers are exploring herbal remedies as potential alternatives. C-Phycocyanin, an extract from Spirulina platensis, shows promise due to its antioxidant and anti-inflammatory properties. Considering the recent reports, the current study was designed to develop a disease model of PD in mice through induction of Diabetes Mellitus using Streptozotocin which can lead to insulin resistance and cause number of PD symptoms. The study also explored the neuroprotective role of C-PC and its possible mechanism of action against Rotenone induced Parkinson’s disease model in mice. Objectives: In this study, we aimed to develop and validate a Parkinson's disease mice model induced by Streptozotocin. The model was used to evaluate the effectiveness of C-Phycocyanin against a rotenone-induced Parkinson's disease model in mice. Additionally, we aimed to investigate the underlying mechanism of action of C-Phycocyanin in this model. By developing this model and studying the effects of C-Phycocyanin, we aimed to contribute to a better understanding of Parkinson's disease and explore potential therapeutic interventions. Materials and Methods: To develop the STZ induced PD model, C57/BL6 mice were separated into three groups, with ten animals in each group: NC: Normal Control, Rotenone (50mg/kg p.o), and Streptozotocin (100mg/kg). The behavioural paradigms were conducted such as Rotarod test, Y-Maze test, Hang test, Beam walk test. Baseline behavioural studies were performed and conducted at alternative weeks respectively. The study was carried out for 6 weeks with administration of Rotenone (50mg/kg) as inducing agent in Rotenone group followed with STZ (100mg/kg) in third group. At the end of 5th week, STZ (100mg/kg i.p.) was administered for induction of chronic diabetes and after 1 day of administration, glucose estimation was performed. At the end of 6th week, under light ether anaesthesia, blood from the retro-orbital plexus was collected in centrifuge tubes, animals were sacrificed and whole brain, hippocampus, striatum, cortex were collected. Oxidative parameters such as GSH and MDA were measured from serum and tissue samples. Neuroprotective parameters such as α-synuclein, AKT, AMPK, PI3K, BDNF, GSK-3β, GLP1, NF-κB were measured from tissue homogenate using ELISA kits. Histopathology analysis such as H and E stain and Nissl stain were performed for neuronal shrinkage, inflamed cells and loss of dopaminergic neurons. To study the pharmacological actions of C-PC against rotenone induced PD in mice, the ICR mice were divided into four groups of eight animals NC: Normal Control, Rotenone (50mg/kg, p.o), Rotenone (50mg/kg, p.o) + C-Phycocyanin (50mg/kg, i.p), C-Phycocyanin (50mg/kg, i.p). Various behavioural paradigms such as Rotarod test, Y-Maze test, Hang test, Beam walk test were performed. All the animals were kept according to their groups. Baseline behavioural studies were performed and conducted at alternative weeks respectively. The study was carried out for 28 days with administration of Rotenone (50mg/kg) as inducing agent in Rotenone group and Rotenone + C-Phycocyanin group. C-Phycocyanin (50mg/kg, i.p) was given as a treatment drug in Rotenone + C-Phycocyanin and C-phycocyanin group. At the end of 4th week, under light ether anaesthesia, blood from the retro-orbital plexus was collected in centrifuge tubes, animals were sacrificed and whole brain, hippocampus, striatum, cortex were collected. Oxidative parameters such as GSH and MDA were measured from serum and tissue samples. Neuroprotective parameters such as α-synuclein, AKT, AMPK, BDNF, NF-κB were measured from tissue homogenate using ELISA kits. Histopathology analysis such as H and E stain and Nissl stain were performed for neuronal shrinkage, inflamed cells and loss of dopaminergic neurons. Results: Development of STZ induced PD Model: Our study demonstrated that a high-fat diet combined with a single dose of STZ (100mg/kg i.p.) induced PD-like symptoms. The STZ model exhibited impaired motor coordination in the rotarod test and reduced grip strength in the hang test after 6 weeks. The beam walk test did not yield significant results. STZ induction led to elevated blood glucose levels, indicating signs of Type 2 diabetes. Inflammation was associated with increased oxidative stress, as evidenced by decreased GSH levels and slightly elevated MDA levels. Higher levels of alpha-synuclein and BDNF were observed, contributing to dopaminergic neuron loss, mitochondrial dysfunction, and reduced cell growth. Reduced levels of AKT and PI3K were observed, leading to cell apoptosis, decreased cell survival and growth, and impaired neurogenesis. The STZ-induced PD group showed lower GLP-1 levels, indicating mitochondrial dysfunction. Increased GSK-3beta levels were linked to neuronal death. Elevated NF-κB and AMPK levels were observed, causing cell inflammation, reduced neuronal loss, and altered antioxidant activity. Histopathological analysis revealed decreased cell size, cell number, and inflammation, while Nissl stain showed dopaminergic neuron loss and neuronal shrinkage Efficacy of C-PC against Rotenone induced PD: Parkinson's disease is induced by Rotenone, a pesticide that causes neuronal dysfunction, mitochondrial impairment, and motor coordination problems. Treatment with C-Phycocyanin (C-PC) improved muscle coordination and motor function in the rotarod test. Grip strength and cognitive abilities, assessed by the hang test and Y-Maze, were enhanced with C-PC treatment. No significant results were observed in the beam walk test. C-PC exhibited antioxidant properties by increasing levels of GSH and reducing inflammation, as indicated by a slight decrease in MDA levels. C-PC also showed positive effects on alpha-synuclein and BDNF levels, preserving dopaminergic neurons and preventing Lewy body formation. AKT and AMPK activity improved with C-PC, enhancing metabolism, cell growth, and survival. C-PC increased AMPK levels, promoting mitochondrial function, cellular antioxidant capacity, and α-synuclein clearance through autophagy. C-PC reduced NF-κB levels, preventing cell inflammation. Histopathological analysis revealed intact neuronal structures, pyknotic neurons, and signs of inflammation in Rotenone-induced animals. After C-PC treatment, neuronal density increased, inflammation decreased, and dopaminergic neurons were restored. Conclusion: Our study suggests that the Streptozotocin-induced Parkinson's disease (PD) model exhibited delayed neurobehavioral signs due to diabetes induction. STZ administration reduced GSH levels and increased MDA levels, leading to PD symptoms in mice. In comparison to the Rotenone model, STZ resulted in significant degradation of neuroprotective markers such as alpha-synuclein, BDNF, AKT, and NF-κB. Treatment with C-Phycocyanin (C-PC) improved motor coordination, working memory, and spatial learning abilities. C-PC also reduced GSH levels and increased MDA levels. Neuroprotective and histopathological analysis showed that C-PC inhibited dopaminergic neuronal loss, reduced inflammation, and demonstrated a protective effect against the Rotenone-induced PD model in mice