| Parkinson’s disease(PD) is a common neurodegenerative disease that mianly damages the motor function of patients, and which additionally influences the execution, attention, memory and the language features of patients throughout the course of disease. The main pathological character of PD is the degeneration of dopaminergic neurons in substantia nigra. Up to now, the pathogenesis of PD is unclear yet, but it is probably related to the oxidative stress, mitochondrial dysfunction, genetic mutations, immune inflammatory reaction and toxic injury etc. Apoptosis is thought to be the convergent point that leads to the loss of dopaminergic neurons. Mitochondria play an important role in the process of apoptosis. Therefore, mitochondria maybe an effective target to prevent or delay PD by increasing mitochondrial biogenesis, improving mitochondrial function and reducing the formation of free radicals.Peroxisome proliferator-activated receptor gamma(PPARγ) is an nuclear receptor, whose signal pathway after being activated could protect the central nervous system neurons by enhancing the mitochondrial biogenesis and the metabolism, the anti-inflammatory, the resistance oxidation damage and the anti-apoptosis etc. 2,4-Thiazolidinedione(2,4-TZD), which can effectively activate the signal pathway of PPARγ, is the agonist of PPARγ. Researches have shown that PPARγ agonists thiazolidinediones(TZDs) can obviously improve the rats’ symptoms of PD injured by MPTP and protect the dopaminergic neurons. The effect and mechanism of thiazolidinediones(TZDs) in vitro cell model of PD need to be further studied.In this study, a vitro cell model of PD was duplicated by the classical method of 1-methyl-4-phenyl pyridineion(MPP~+) damaging SH-SY5 Y cells(dopaminergic cells). MTS assay, Hoechst 33342 staining, FJC staining and Western Blot were used to explore the effect of 2,4-TZD on SH-SY5 Y cells damaged by MPP~+, and make that whether 2,4-TZD can improve the mitochondrial function, reduce apoptosis and protect the dopamine neurons by regulating the expression of PGC-1α and its downstream signaling molecule.The experimental results are as follows:1. The survival rate of SH-SY5 Y was reduced to 48% with the concentration of MPP~+ 1 mmol/L at 24 h. The model in vitro was successfully duplicated.2. There was no difference in cell survival rate among blank control group and other different concentration groups while 2,4-TZD put acts on the SH-SY5 Y cells individually(P>0.05).3. MTS assay was used to detect the effect of the co-incubation of 2,4-TZD and MPP~+ on SH-SY5 Y cells for 24 h and we found that, 0.01, 0.1, 1, 10 μmol/L 2,4-TZD intervention groups could significantly increased the cell survival rate(P<0.05), and 0.01, 0.1, 1μmol/L 2,4-TZD three lower concentrations have better results than 10 μmol/L(P<0.01).4. We examined the effect of 2,4-TZD on the SH-SY5 Y cells apoptotic rate imparide by MPP~+ was discovered that, those intervention groups with 0.01, 0.1, 1,10 μmol/L 2,4-TZD respectively all presented a remarkable reduction in the apoptotic rates of cells(P<0.01). Besides, the first three groups with 0.01, 0.1, 1 μmol/L 2,4-TZD respectively demostrated better results than the group with 10 μmol/L 2,4- TZD.5. The results of Western Blot method have detected the expression levels of function-related proteins of mitochondrial(PGC-1α, NRF1, NRF2) and showed that, the expression of PGC-1α in 0.1, 1 μmol/L 2,4-TZD intervention groups significantly increased compared with MPP~+ group(P<0.05); and the expression of NRF1, NRF2 in 0.01, 0.1, 1 μmol/L 2,4-TZD intervention groups significantly increased compared with MPP~+ group(P<0.05).6. Western Blot was used to detect the expression of mitochondrial fission and fusion related proteins(Fis1, Mfn2) and we found that: the ratio of Fis1/Mfn2 significantly increased after damaged by MPP~+, and the ratio of Fis1/Mfn2 in 0.01, 0.1 μmol/L 2,4-TZD intervention groups reduced. But the ratio of Fis1/Mfn2 in 1, 10 μmol/L 2,4-TZD intervention groups significantly increased comparing with 0.01, 0.1 μmol/L 2,4-TZD intervention groups.7. The result of the expression of apoptotic and survival proteins(Bax, Bcl-2, ERK1/2) detected by Western Blot, which displayed that, the expression of Bax in intervention groups with 0.01, 0.1 μmol/L 2,4-TZD respectively showed a significant reduction versus the MPP~+ group(P<0.05); and the expression of Bcl-2 in intervention groups with 0.01, 0.1, 1 μmol/L 2,4-TZD respectively displayed a significant increase versus the MPP~+ group(P<0.05). The expression of ERK1/2 in intervention groups with 0.1, 1 μmol/L 2.4-TZD respectively presented a significant increase versus the MPP~+ group(P<0.05).We can draw the conclusions through analyzing the above results:1. 2,4-TZD can increase the cell survival rate, improve the cell state and protect dopaminergic cells SH-SY5 Y from damaging by MPP~+.2. 2,4-TZD can protect SH-SY5 Y cells from damaging may by activating PGC-1α and its downstream signals NRF1, NRF2, which then regulating the expression of mitochondrial fission protein Fis1 and fusion protein Mfn2 and maintaining the homeostasis of mitochondrial fission and fusion as well.3. The protection of 2,4-TZD for the SH-SY5 Y neurons through reducing the expression of pro-apoptotic protein Bax, and also increasing the expression of anti-apoptotic Bcl-2 protein and survival protein ERK1/2.The above results indicate that 2,4-TZD can significantly protect SH-SY5 Y cells from MPP~+ damaging. 2,4-TZD reduces apoptosis may be increasing PGC-1α and its downstream signals NRF1, NRF2, then promoting mitochondrial biogenesis, improving mitochondrial function. This study can provide basic research data for 2,4-TZD as the potential drug in prevention and treatment of PD. |
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