Neuroprotective Effect Of Bilirubin On Parkinson’s Disease

Parkinson’s disease （PD） is clinically characterized by tremor, rigid, bradykinesia，posture disfunction，with or without autonomic nerve disfunction. Nowdays, the incidencerate and numbers of the disease increased with the progress of population aging in ourcountry. The total number of patients with PD has exceeded2,000,000. PD is bound toinfluence the life quality of aged people and pose spirit and economic burden to patientsthemselves, to families and to the society. Therefore, it is important to study on pathologicmechanism of PD and pursue new therapeutic strategy for PD. Pathological feature in PDis the degradation of dopaminergic neurons, for which the reason is still unclear. Howeverthe popular and widely acknowledged standpoint is that oxidative stress and mitochondrialdisfunction develop considerable effect on PD。In these regards, the effect of antioxidantsin PD and PD therapy has attracted people’s attention and become hot in research area.Bilirubin, traditionally regarded as a toxic metabolic waste, is the end product ofheme. More and more researchs indicated that bilirubin, as the only endogenous andlipophilia antioxidant, has powerful anti-oxidant and anti-inflamation effect. Therelationship between bilirubin and coronary artery disease and diabetes mellitus has beenconfirmed. Bilirubin neuroprotective effect on neurons and hippocampus had been alsoreported, but its effect on DA and the relation between bilirubin and PD have not yet beenresearch on.This study was designed to investigate the relationship between bilrubin and PDincidence by retrospective analysis of clinical case data, on the basis of which PD cellmodel was further established to evaluate the effect and the mechanisms of bilirubin on PDjust on cellular level. Part oneStudy on serum bilirubin, uric aci and lipid levels in patientswith Parkinson’s diseaseObjective: To detect serum bilirubin （IBIL）, uric acid （UA） and lipidsconcentrations of patients with Parkinson’s disease and discuss possible relationshipbetween them.Methods: Serum IBIL, UA and lipids concentrations were measured and recorded in250patients with Parkinson’s disease who were identified in Xuzhozu Central Hospitalfrom2006-2012.259age and gender matched healthy people identified in the medicalexamination center of the hospital were randomized selected as controls．All data wereanalyzed with SPSS17.0software.Results: IBIL and UA concentrations in PD group were significantly lower thancontrol （P＜0.05）. In PD group further, IBIL and UA concentrations of female subgroupwere significantly lower than male subgroup （P＜0.05）. However there were nodifferences among age subgroups and H&Y subgroups（P＞0.05）. IBIL concentrationpositively correlated with UA concentration only in control group（r=0.229，P＜0.01）, butnot in PD group（r=-0.032，P＞0.05）. TCH and LDL-C concentrations in PD group weresignificantly lower than control （P＜0.05）. And in PD group further, TCH and LDL-Cconcentrations of female subgroup were significantly higher than male subgroup （P＜0.05）. HDL-C and TG concentrations had no differences between PD group and control（P＞0.05）. TCH, LDL-C, HDL-C and TG all had no differences among age subgroupsand H&Y subgroups（P＞0.05）.Conclusions: Serum IBIL and UA concentrations lowered in PD patients and evenlower in female PD patients than male. IBIL positively correlated with UA in control butsuch relationship was disrupted in PD group. Serum TCH and LDL-C concentrations alsolowered in PD patients but even lower in male PD patients than female. Respectively, IBIL,UA, TCH and LDL-C had no differences among age subgroups and H&Y subgroups. Part TwoBilirubin pretratment alleviate the impairment of6-OHDA onSH-SY5Y cellsObjective: to confirm whether bilirubin pretreatment alleviates the impairment of6-OHDA on SH-SH5Y cells.Methods: PD cell models were established in vitro with6-OHDA and SH-SY5Y cells.Various concentrations of6-OHDA or bilirubin were used to treat with SH-SY5Y cellsrespectively, and at different endpoints the changes of cell morphous, MTT reduction andLDH release were observed and recorded. Data were analyzed with SPSS17.0software.Results：Being exposed to25μM、50μM、100μM，150μM，200μM6-OHDA for24h,SH-SY5Y cells morphous changed, away from their original shape, with MTT reductiondecreasing and LDH release increasing in a concentration dependent manner（P＜0.05）,which indicated that cell viability was degraded and cell membrane integrity wasdestroyed.Being exposed to30μM bilirubin for4h，SH-SY5Y cells MTT reduction wassignificantly lower than contro（lP＜0.05）；Being exposed to5μM and10μM bilirubin for24h，cell MTT reduction increased and LDH release decreased（P＜0.05, VS control&4h）.Being exposed to1μM、5μM and10μM bilirubin for48h, cell MTT reduction increasedand LDH release decreased significantly（P＜0.05, VS control&24h）, especially to10μM bilirubin. While being exposed to30μM bilirubin for48h, cell MTT reduction decreasedand LDH release increased significantly（P＜0.05, VS control&24h）. The results indicatedthat1μM、5μM and10μM bilirubin had proliferative effect on SH-SY5Y cells atendpoints of24h, but30μM bilirubin had toxic effect on SH-SY5Y cells as early as4h.As to pretreatment experiment, being exposed to1μM、5μM、10μM、15μM、30μMbilirubin for4h, and then treated with100μM6-OHDA for24h, SH-SY5Y cells MTTreduction decreased and LDH release increased significantly in both un-pretreated group and pretreatment group（P＜0.05, VS control）; but MTT reduction increased and LDHrelease decreased significantly in pretreated group when compared with un-pretreatedgroup（P＜0.05, pretreated VS un-pretreated）.Conclusions:6-OHDA had toxic effect on SH-SY5Y cells in a concentration dependentmanner. Pretreatment of10μM bilirubin could alleviate the impairment of6-OHDA onSH-SY5Y cells. Part ThreeNeuroprotective mechanism of bilirubin agaist the impairmentof6-OHDA on SH-SY5Y cellsObjectives: To investigate the mechanisms of bilirubin against the impairment of6-OHDA on SH-SY5Y cells.Methods: Being exposed to10μM bilirubin for4h, and then treated with100μM6-OHDA for24h, SH-SY5Y cells were collected for further detection. ROS and MDAlevels, SOD activity and GSH level were measured. Caspase-3acitvity and apoptosis cellratio were detected. There was no bilirubin pretreatment and no6-OHDA treatment incontrol group, and only no bilirubin pretreatment in un-pretreated group.Results: In control group, un-pretreatment group and pretreatment group respectively,ROS levels were:1±0.23、2.56±0.75and1.69±0.35. MDA levels were:0.56±0.24nmol/mgprot、2.07±0.32nmol/mgprot and1.39±0.23nmol/mgprot. SOD levels were:24.35±1.27U/L、10.53±1.35U/L and16.15±1.72U/L. GSH levels were:10.19±1.67μmol/g、2.98±0.75μmol/g and5.79±1.30μmol/g. Caspase-3levels were:1±0.24、1.89±0.35and1.46±0.22. Apoptosis cell ratios were:4.47±0.35%、10.74±0.95%and7.05±0.56%。Compared with control group, in both pretreated and un-pretreated group,ROS and MDA levels increased significantly（P＜0.05）, SOD activity and GSH level weredecreased significantly（P＜0.05）, Caspase-3activity enhanced and apoptosis cellsincreased（P＜0.05）。Compared with un-pretreated group, in pretreated group, ROS and MDA levels decreased significantly（P＜0.05）, SOD activity and GSH level wereincreased significantly（P＜0.05）, Caspase-3activity weakened and apoptosis cellsdecreased（P＜0.05.Conclusions:6-OHDA induction led to an increase of ROS and MDA levels,decrease of SOD activity and GSH level, enhancement of Caspase-3activity and apoptosiscells ratio（P＜0.05）. However,10μM bilirubin pretreatment could obviously alleviatethese toxic effect, by produing a decrease of ROS and MDA levels, a decrease ofCaspase-3activity and apoptosis cells ratio, but a increase of SOD activity and GSH level, Overall Conclusions1. Serum IBIL and UA levels lowered in PD patients, suggesting that bilirubin might alsobe involved in PD development and progress, just as UA did.2.6-OHDA had toxic effect on SH-SY5Y cells in a concentration dependentmanner.30μM bilirubin had toxic effect on cells and pretreatment with10μM bilirubincould alleviate the impairment of6-OHDA on SH-SY5Y cells. Therefore, bilirubin hadneruoprotective effect on PD cell modle.3. Bilirubin could alleviate the toxic effect of6-OHDA by reducing ROS and MDA level,degrading Caspase-3activity and apoptosis cells, but enhancing SOD activity and GSHlevel.4. Bilirubin accomplished its neuroprotective effect by modulating the balance betweenoxidation and anti-oxidation, and inhibition to lipid peroxidation might be the primarymechanism for bilirubin neuroprotection on PD.