The Preliminary Study Of Protective Effect Of Thioredoxin Reductase 1 In Parkinson’s Disease

Objective:Parkinson’s Disease (PD) is a common degenerative disease. At present, the complete etiology of PD is unclear, but it is widely believed that oxidative stress and apoptosis play important roles in PD pathology. Antioxidant and anti-apoptosis therapy have been the effective methods for PD treatment. Thioredoxin Reductase (TR) is a NADPH-dependent antioxidant enzyme, its main function is to catalyze the oxidized Thioredoxin (Trx) to reduced one and has antioxidant action. The members of TR family include TR1, TR2 and TR3. Althrough TR1 is the main type of TR, the role of TR1 in the brain of PD is unclear. Our research is divided into two parts. The expression levels of TR1 and activity of TR in the midbrain and substantia nigra pars compacta (SNc) of PD mouse model induced by MPTP and the TR1 protein expression in PD cell model induced by MPP+ were investigated in Part One. In Part Two, the eukaryotic expression vector of rat TR1 was constructed and transfected into PC 12 cells to observe whether TR1 has protective effect on PD cell model.Methods:In Part One, C57BL/6 mice were randomly divided into two groups, the normal group and the MPTP group. Mice in the MPTP group were injected i.p.4 times (at 3h intervals over 1 day) with 20 mg/kg MPTP hydrochloride dissolved in saline. Mice in the normal group received an equivalent volume of saline. Bahavioral change was measured and Tyrosine hydroxylase (TH) in the SNc was evaluated by immunohistochemical method. Then the levels of GSH and MDA and the activities of SOD and TR in the midbrain were detected by using the colorimetric method. The levels of TR1 mRNA and protein in the midbrain were determined by employing real-time RT-PCR and western blot analysis, respectively. TR1 positive cell number in the SNc was evaluated by using immunohistochemical method. In addition, TR1 protein expression in PD cell model induced by MPP+ was evaluated by western blot.In order to better study the role of TR1 in PD, the eukaryotic expression vector of TR1 was constructed in Part Two. Firstly, total RNA of PC12 cells was isolated and transcribed to cDNA using oligo(dT) primer. Then the full length of rat TR1 cDNA fragment was amplified by PCR. The full length of rat TR1 cDNA fragment and plasmid pcDNA 3.1/myc-His (-)-3 FLAG-IRES-hrGFP were digested by EcoRI and BamHI. The recombinant eukaryotic expression vector of rat TR1 was carried out. The monoclone was selected and submitted to get the sequence of the recombinant eukaryotic expression vector of rat TR1.After sequencing, the recombinant was transfected into PC 12 cells. The transfection efficiency was observed under fluorescence microscope and the expression level of TR1 protein in PC12 cells was detected by western blot, and the survival rate of PC12 cells was detected by MTT method.Results:In Part One, we found that muscle tension had become weak in behavior testing, the number of tyrosine hydroxylase (TH) positive neurons reduced in SNc, which suggesting the successful PD mouse model. Then oxidative damage index of correlation GSH and SOD decreased and MDA increased in the MPTP groups when compared with the normal groups. It was shown that the levels of TR1 mRNA and protein were decreased in MPTP-challenged mice compared to normal mice. Further, a significant reduced TR activity was found in the MPTP-treated mice compared with normal mice. Consistent with these results, the numbers of the TR1 immunopositive neurons in the SNc of MPTP group were significantly lower when compared to those of normal group. In PD cell model, TR1 protein expression was also decreased.In Part Two, rat wild type TRl gene cDNA sequence was successfully proved by sequencing and compared with GeneBank, then we transfected TR1 recombinant into PC 12 cells. We detected transfection efficiency of 50% by GFP protein expression under the fluorescence microscope, and the relative expression level of TR1 protein was increased obviously in PC12 cells transfected TR1 recombinant. Additionally, we demonstrated that TR1 recombinant could against the PC12 cells injury induced by MPP+ and significantly increase PC12 cells survival rate.Conclusion:The relative expression levels of TR1 were decreased in the PD mouse model induced by MPTP and PD cell model induced by MPP+. pcDNA.3.1/myc-His(-)B-3×FLAG-IRES-hrGFP-TRl recombinant was successfully constructed and expressed in PC 12 cells and it has a protective effect on PC12 cells against MPP+ injury.