The Preliminary Study Of Neuroprotective Effect By Leptin In Parkinson’s Disease

Pathological changes in the brain of Parkinson’s disease (PD) are substantia nigradopaminergic neuronal degeneration, death, loss, substantia nigra-striatal dopaminergicsystem dysfunction, and α-synuclein and ubiquitin staining positive cytoplasmicinclusion bodies formation showed in the remaining neurons. Thus to find an effectiveneuroprotective factor, which is to intervent the main part of Parkinson’s disease, toreduce nerve damage and to restore nerve function, has been one research focus.Leptin is a central regulator of energy metabolism, and plays an important functionin inflammation and wound healing. Recent studies about its neuroprotective effectsattracted widespread concern, but researchers still knew little about the mechanism ofits action.In order to understand the role of leptin and its pathophysiological mechanisms inPD, this study intends to the relation between leptin and PD by PD patients bloodanalysis and the regulation of leptin on Parkinson’s disease and its mechanism byestablishing6-OHDA-induced C57BL/6J mouse model of Parkinson’s diseases and6-OHDA and rotenone-induced dopaminergic-like SH-SY5Y cells model of Parkinson’sdisease.The topics include the following three parts of work:1. The changes of serum leptin, its receptor and oxidative stress of Parkinson’sdisease compared with in the non-PD control groupObjectives: To investigate the relationship between the serum levels of leptin,leptinreceptor and indicators of oxidation and antioxidant and Parkinson’s disease.Methods:1Leptin and sObR of healthy people (various age group) were detected;2Leptin and sObR levels and MDA, GSH and SOD levels of Parkinson’s patients andcontrol group (mainly older age groups) were detected;3Leptin and sObR levelsassociated with Parkinson’s disease, age, and gender were analyzed;4MDA, GSH andSOD levels associated with Parkinson’s, leptin and sObR.Results:1With age increasing, leptin levels increased but stabilized at around40years old, while little change in sObR;2In PD patients, relative to control group (older agegroups), MDA and sObR levels increased, but leptin, SOD and GSH levels decreased(P <0.05);3Leptin levels of females were higher than those of males while sObR levelswas reverse (P <0.05);4GSH and SOD levels were positively correlated with leptin andnegatively correlated with sObR while MDA and leptin levels were reverse.Conclusions:1. In PD patients, serum leptin levels were lower while serum sObRlevels were higher and serum levels of oxidative stress were higher.2To maintainhigher serum leptin levels, the Parkinson’s disease risk could be reduced.2. Leptin protecting nigra region in Parkinson’s disesases animal modelsObjectives: Based on Parkinson’s diseases animal models, the substantia nigraregion protected and downstream oxidative stress levels changed by leptin wereobserved. Methods:①6-OHDA-induced Parkinson’s diseases animal models with rightnigra region injury were established. The experiment group included sham group(control group), model group, model+leptin, model+leptin+nanobody group. Whethermodels were successfully established by observing the number of animal’s left rotationafter subcutaneous injection of apomorphine.②Immunohistochemistry detectedmidbrain dopaminergic substantia nigra neurons specific protein tyrosine hydroxylasechange of each group;③Serum MDA, GSH and SOD levels of each group weredetected.Results:1After subcutaneous injection of apomorphine, relative to control group,the left rotation number of model group significantly increased, which was attenuatedby leptin, while after adding nanobody, the rotation number significantly increased.2Relative to control group, the number of dopaminergic in right substantia nigra regionof model group significantly reduced, which was attenuated by leptin, while afteradding nanobody, the number of the dopaminergic neurons in right substantia nigraregion significantly increased.3Relative to control group, serum MDA levels of modelgroup increased while serum GSH and SOD levels of model group decreased, whichwas reversely changed by leptin, while after adding nanobody, leptin function wasattenuated. Conclusions:1Exogenous leptin could reduce the severity of Parkinson’s diseaseand reduce the loss of dopaminergic neurons;2In animal models of Parkinson’sdiseases, leptin could significantly reduce the level of serum MDA and elevated SODand GSH levels, which played a active role in blocking lipid peroxidation, increasingantioxidant activityand maintaining stability in the internal environment.3. The mechanism of leptin to reduce the6-OHDA and rotenone-inducedSH-SY5Y cell injuryObjectives:6-OHDA and rotenone-induced SH-SY5Y cell model of Parkinson’sdisease and neuroprotective mechanisms of leptin were discussed. Methods: SH-SY5Ycells were induced by6-OHDA and rotenone.①The survival and injury rates ofdifferent concentrations of6-OHDA rotenone-induced SH-SY5Y cells were detected byCCK-8and LDH.②CCK-8, LDH, trypan blue and flow cytometry were used todetect the effect of100μmol/L6-OHDA and5mol/L rotenone-induced SH-SY5Y cellsurvival, injury, death, and apoptosis by leptin.③Fluorescent probes detects the effectof6-OHDA and rotenone-induced ATP and ROS, which leptin changed. Western blotand Immunofluorescence detected cleaved caspase-3levels changed by leptin.④After nanobody added, the changes of SOD, MDA and GSH (oxidation resistance andoxidation resistance of these indicators) in relevant cell lysates changes were detected.⑤CCK-8tested the effect of6-OHDA and rotenone-induced injury reduction for leptinby AG490, nanobody, LY294002, compound C, U0126and ZnPP and the effect of LiCland CoPP addition in the model.⑥After adding LY294002, compound C and U0126,western blot detected the apoptosis-related proteins Bcl-2, Bax, as well as relatedpathway molecules p-Akt, p-ERK1/2, p-AMPK, p-GSK3β, HO-1. Western blot andRT-PCR detected Parkinson’ diseases-associated protein α-synuclein. Western blotdectected Bcl-2, Bax and HO-1levels of LiCl and Copp addition in model.Results:1Leptin improved survival of SH-SY5Y cells, reduced the damage andapoptosis rate;2Leptin improved the increasing of ATP, reduced the level of ROS,which was blocked by nanobody partialy.3Blockers adding reversed MDA redcuction,SOD and GSH increasing caused by leptin;4After appropriate blocking agent added, the protective effect of leptin were attenuated;5Leptin increased the expression ofBcl-2and HO-1, reduced the expression of caspase-3activity, Bax and α-synuclein, andimproved the levels of p-Akt, p-ERK1/2, p-AMPK and p-GSK3β. Antagonist addingreversed the effects of leptin and relevant protein blockers adding reduced the p-GSK3βlevels and reversed Bcl-2and HO-1increasing expression of leptin, and increased Baxreduction of leptin;.Conclusions:1Based on cell model of Parkinson’s diseases, leptin couldreducesthe damage;2Leptin could protect the cell model by converging on GSK-3βthrough Akt, ERK and AMPK pathway, then participated in the downstream. Thedownstream protection is mainly to increase HO-1expression, SOD, GSH and ATPlevels and reduce ROS, MDA and LDH levels so that leptin could reduce apoptosislevels and cell injury.