Investigation of Anti-Diabetic Agents in Hyperglycemic Brain Insult and Associated Cognitive Decline

Abstract

Diabetes is a chronic metabolic disorder and affects the multiple system of human body. The epidemiologic number of diabetes associated systemic complications are more in number than any other disease in the world population. Diabetes is associated with variety of set of morbid complications like ischemia, vascular dementia and cognitive decline. The diabetics have impaired metabolic functions which may lead to cerebrovascular, cardiovascular and neuropsychiatric disorders. Since past few decades, research is on going with some class of antidiabetic agents such as biguanides, sulfonylureas, thiazolidinediones for their neuroprotective properties, but nowork has been reported with GLP-1 analogues, α-glucosidase inhibitors, SGL2 inhibitors and DPP4 inhibitors for diabetes associated brain insult and cognitive decline.Therefore, the objective of our study wasto analyses such anti-diabetic class of drugs for neuroprotective action who exert a strict hyperglycemic control. We have selected voglibose, saxagliptin, repaglinide and dapagliflozin and screened using in-silico molecular docking with proteins which are linked through various mechanisms associated with hyperglycemia and neurodegeneration.Further, we performed in-vitro cell line studies for their neuroprotective effect. Thereafter, we aimed to develop anin-vitromodel of Zebrafishwhich can re-produce and mimic the action and pathophysiology of strokesimilartohuman being. Zebrafish exerts very similar genetic similarities and also can be useful for diabetes associated stroke research. Therefore, we developed a chemical induced stroke model which can induce similar pattern and pathophysiology of stroke comparing to rodents. Moreover, we also screened all anti-diabetic drugs anti-stroke potential through this induction model. Add on, we observed cognitive improvement in all-anti diabetic drug treated fishes. After this evaluation, we evaluated diabetes associated stress and cognitive decline through UCMS induced stress model in adult zebrafish. The adult fishes were treated with 114 mMol D-glucose for 14 days to induced diabetes and stress was induced with UCMS protocol. The fishes were underwent behavioral paradigm to access the neuroprotection offered by various anti-diabetic agents over post treatment. After UCMS protocol, we evaluated neuroprotective profile of anti-diabetic agents through alcohol induced neurotoxicity and cognitive decline. Diabetic alcoholics are always at a great risk of developing dementia, worsen metabolic disturbances and worsen cognitive profile. Therefore, we induced alcohol induced neurotoxicity into high fat diet induced diabetic rats. We also evaluated brain damage and lesion through H&E staining and GFAP immunohistochemistry. We observed a great hippocampus gliosis, more number of dead neurons, activated astrocytes and red stained astrocytes. After assessment of anti-diabetic agent’s neuroprotective profile, we evaluated itsefficacy and therapeutic effectiveness in MCAo induced diabetic rats. We screened the antistroke potential of anti-diabetic agents through middle cerebral artery occlusion model by administrating pre and post ischemic treatment. We evaluated the effects of anti-diabetic agents on neurological score, neurobehavioral changes, biochemical biomarker changes, anti-oxidant activity, effects over neurotransmitters and also over gene expression in ischemic rats’ pre and post operatively. Then, UCMS study in mice was carried to evaluate the role of anti-diabetic agents in diabetes associated depression and stress. The mice were induced diabetes with dexamethasone through s.c injections for 14 days 1 mg/kg s.c. The mice underwent 21 days UCMS stress protocol to induced stress into them and they were post treated with anti-diabetic agents for two weeks to evaluate the anti-depressant or cognitive enhancer therapeutic efficacy of anti-diabetic agents. The diabetic individuals often go with surgical procedures and anesthesia exposures which may contribute triggering the neurodegeneration profile into them. So that we aimed to evaluate the neuroprotective potential of anti-diabetic agents over anesthesia exposure in diabetic rats we assessed the dexamethanose induced diabetic rats through chronic anesthesia exposure and post treated them with selected anti-diabetic agents.From the docking study, we found a good docking score and docking energy with these four drugs and selected proteins associated with neurodegeneration.The IC50 values for voglibose, saxagliptin, repaglinide and dapagliflozin in SK-N-SH cell lines were found to be 7.2 μM, 8.4 μM, 6.2 μM and 18.6 μM respectively in low glucose media and 7.6 μM, 8.66 μM, 6.9 μM and 15.6 μM in high glucose media respectively. All anti-diabetic agents showed neuroprotective and cytoprotective actions in MTT assay. We developed global ischemia in adult zebrafish with 3μgm endothelin-1 by oral administration in which we observed a significant brain damage and global ischemia in ET-1 induced adult zebrafish. We got voglibose and saxagliptin exerting neuroprotection over ischemic brain insult. We found all selected anti-diabetic agent offer a mild to moderate neuroprotection and improved cognition. Among all, saxagliptin, voglibose and dapagliflozin were found very effective in improving cognitive decline. We observed a great cognitive decline and alcohol withdrawal in diabetic alcoholic rats. Post treatment with antidiabetic agents significantly improved all biochemical biomarkers, cardiac markers, lipid profile and also cognition by post treatment in diabetic alcoholic rats. The anti-diabetic drug treatment with voglibose, saxagliptin and dapagliflozin significantly reduced overall neurotoxicity in alcoholic diabetic rats and improved learning, memory and cognition. The most effective antidiabetic was found to be saxagliptin>voglibose>dapagliflozin>repaglinide. We found voglibose and saxagliptin reduced the risk factors related to ischemia in pre-stroke treatment andreduced the post stroke brain damage, improves cognition and also reduced recurrent stroke bio-markers. Dapagliflozin and repaglinide have shown very strict glucose control and very positive effects over cardiac risk factors in pre-stroke treatments. Both the drugs significantly reduce the cardiac risk factors related to hyperglycemic brain damage while administered pre-operatively. Post treatment with both drugs greatly improves cognitive profile but having poor anti-stroke potential in recovering ischemic brain damage. Upon, GFAP staining of various brain sections, it was found that pre and post exposure of saxagliptin and voglibose significantly reduced the brain damage and reduced brain lesions which contributes to stroke associated cognitive demur. Moreover, voglibose and saxagliptin improves gene expressions of stroke related genes like TGF-beta and VEFG. Both drugs also significantly down-regulate inflammatory gene expressions of TNF-alpha and MMP-9. Where, dapagliflozin and repaglinide have poor control over gene regulation of stroke associated genes over pre and post treatments in MCAo induced diabetic rats. The cerebrospinal fluid proteomic analysis of ischemic diabetic rats through SDS-Page of these pre and post stroke treatments revealed a great role of saxagliptin and voglibose in reducing E-cadherin, TKRc and MMP-9 protein expressions which significantly contributes to ischemia and further brain damage. Dapagliflozin contributes to mild to moderate reduction of such protein expressions compared to voglibose and saxagliptin. We found, saxagliptin and dapagliflozin exert a great cognitive enhancer and anti-depressant profile over diabetic stress condition. The therapeutic potential in improving cognition and reducing depressive like behavior from anti-diabetic treatment was found to be saxagliptin>dapagliflozin>voglibose>repaglinide. We observed a very good neuroprotective action in post treatment with saxagliptin, voglibose and dapagliflozin treated chronic anesthesia exposed rats. Repaglinide was found to be least effective in improving overall neuroprotective profile in anesthesia exposed diabetic rats.