IGF-1 Protects Dopaminergic Neurons From L-DOPA-induced Toxicity Via The JAK/STAT Pathway

Recent studies showed that diseases of the nervous system such as neural system degeneration, brain ischemia, cerebral hemorrhage, epilepsy, brain trauma, anoxia encephalopathy etc have several similar or common physiological and pathological mechanisms. Cell apoptosis or necrosis exists which result in the loss of neurons impacting function. Effective intervention to nerve cell apoptosis process is the key research of neurology, also will be the focus of therapy in the future.Parkinson's disease was first described by James Parkinson in 1817 as a tarditive neural degenerative disease. Clinical characteristics manifest as moving slowly, static tremor, gear like muscle tension and abnormal posture reflection. Pathological changes are dopaminergic neurons degeneration and apoptosis of the nigrostriatal area in the midbrain and the formation of lewy small body. Morbidity in people more than 60 years old reaches to 1-2%. At present the treatment of the disease is dominated in controlling symptoms. The development of the disease can't be hindered or reversed. The shortage of dopaminergic neurons of the nigrostriatal area in the midbrain lead to the insufficient of dopamine in the body. Exogenous supplement of levodopa (levodopa, L-dopa) is an important treatment strategy of Parkinson's disease. However patient who intake levodopa for a long term will have treatment effective droped. About 30% to 50% of patients using the drug for five years or longer have symptoms fluctuations.Insulin-like growth factor 1 is a non-selective neurotrophic factor. Its receptor widely distributed in various human body organs, such as dopaminergic neurons in the midbrain nigrostriatal area. Insulin-like growth factor-1 plays a role in neurons through activating multiple signal transduction pathways. JAK/STAT signal transduction pathway acts in stress reaction, which broadly participates in processes such as cell proliferation, differentiation mature, apoptosis and immune modification. It is one of the important ways that cytokines do signal transduction. Studies confirmed that insulin-like growth factor-1 can also functions through activating JAK/STAT pathway. But there were inconsistencies in different diseases. Therefore, whether JAK and STAT proteins can be upregulated or activated in the apoptosis or necrosis of neural cells and what is the role of JAK/STAT? This paper launched the following research works.PartⅠThe establishment of PC12 cell apoptosis model induced by L-dopa and the determination of concentrations of IGF-1 treated.OBJECTIVE:1. To explore the concentrations of levodopa in inducing the cell apoptosis of PC12 cell.2. To determine the concentrations of IGF-1 in protecting the cell apoptosis of PC12 cell.METHODS:MTT assay was used to detect the survival rate of PC 12 cell;RESULTS:1. The determination of the concentration of levodopa causing 50% PC 12 cell apoptosis: Different concentrations (10,20,50,100,150 and 200μmol/L) of L-dopa were used to treat cells for 24 hours, then MTT method was used to detect the cell vitality. Outcomes showed that the cell survival rate of the 10μmol/L group was similar as the blank control group, (96.77±0.55)% vs(97.56±0.52)%. Along with the increase of drug concentrations the cell vitality of PC 12 cell gradually declined, which was most obvious between 100 and 200μmol/L. The cell vitality was (29.86±2.28)% in 200μmol/L.According to the experimental results, we chosed the concentration of levodopa as 150umol/L, and damaging time as 24 hours for the next work.2. The determination of the concentration of insulin-like growth factor 1 in protecting PC 12 cell apoptosis.In order to detect the protection value of insulin like growth factor-1 in PC12 cell, we used the conclusions from the previous research.150 umol/L of L-dopa was used to treat PC12 cell. Different concentrations of IGF-1 (0,25,50,100,150nmol/L) were added respectively. MTT was used to detect the cell vitality. Outcomes showed that the protective action was not obvious when the concentration of IGF-1 was 50nmol/L or below. When concentrations increased, the cell survival rate improved, which was most significant in 100nmol/L group when compared with pure L-dopa group, (80.35±1.64)% vs (62.20±2.03)%. It improved further in 200nmol/l, (83.36±0.96)%.According to the experimental results, we selected the protective concentration of insulin like growth factor-1 as 100nmol/L, treatment time as 24 hours for the next work. CONCLUSION:1. Traeaing the PC 12 cell with L-dopa for 24 hours,The servival rat of cells was obviously decreased,and with the dose increaesing the servival rat was decreaseing significantly.2. 100nmol/L of insulin-like growth factor 1 treating PC 12 cell for 24 hours has protective influence on the apoptosis of PC 12 cell induced by levodopa.Part II The role of JAK/STAT signal pathway in the p rotective effect of IGF-1 in the PC12 cells induced by L-dopaOBJECTIVE:To explore the role of JAK/STAT pathway in the protective impact of IGF-1 inPC 12 cell apoptosis.METHODS:1. Western blot was used to qualitatively and quantitatively analyze various proteins activated by IGF-1;2. Hochest dye was used to detect the apoptosis of PC 12 cell.3. Flow cytometry was used to detect the apoptosis of PC 12 cell.RESULTS:1. No expression of P-JAK2/P-STAT3 protein was found in pure L-dopa treatment group.2. Expression of P-JAK2/P-STAT3 protein was found both in L-dopa plus IGF-1 group and L-dopa plus IGF-1 and AG490 group.3. JAK2 specifical inhibitors AG490 was added 20 minutes before IGF-1 and L-dopa. The expression intensity of P-JAK2/P-STAT3 protein weakened in this group compared to L-dopa plus IGF-1 group. Statistical significant difference in semi-quantitative grayscale value was found between these two groups (P< 0.05). CONCLUSION:1. No activation of JAK2/STAT3 signal pathway was found in PC 12 cell when treating with pure levodopa.2, IGF-1 induced the activation of JAK2/STAT3 signal transduction pathway and has protective effect on the apoptosis of PC 12 cell induced by levodopa.