| Parkinson’s disease (PD) is a central nervous system degenerative disease. Its main pathological feature is the progressive loss of substantia nigra dopamine neurons, further leading to decreased dopamine neurons in the striatal. Marker is the appearance of lewy body. The main clinical manifestations of PD are myotonia, progressive bradykinesia, static tremor, and posture gait disorder and so on. At present,the precise mechanism of this disease is not yet clear. Present study is generally accepted that the PD disease is related to environmental factors, mitochondrial dysfunction,genetic factors, oxidative stress, genetic mutation, neurotoxicity,inflammation and autophage and so on, may also be the result of joint action by multiple factors. PD always occurs in the elderly people, and as the social phenomenon of an aging population, PD have badly hurt on quality of life of themselves and their family. Current treat methods are drug therapy, deep brain stimulation,cell transplantation,and gene therapy,etc. The symptoms and signs of PD can be alleviated by the drugs,which can increase dopamine function. One of the most effective is levodopa. But levodopa appears a series of side effects to limit its wide application. More disappointingly,levodopa might damage neurons and accelerate the apoptosis of neurons for long-term use. So far,we have not yet found effective drugs and methods for the PD. Therefore exploring the mechanisms of the occurrence and development of PD and seeking the best PD treatment strategy are of great significance.More and more studies showed that apoptosis of nigra neurons in PD was related to mediating of a variety of nerve signal transduction pathways, such as JNK, the activation of P53, cell cycle activation, etc. Past researches have shown that tau protein was the common substrate of most known pathways. Since the research has found that the tau protein was important component of the paired helical filaments (PHFs) of neurofibrillary tangles (NFTs) in AD, tau protein was in focus. Tau protein was a kind of protein,which distributed in the neuronal axons, promoted microtubules equipment and stabled associated microtubule. However, the phosphorylation of tau was a kind of pathological changes in the specific sites of human brain, and associated with degenerative disease and dementia. Function of tau protein subjected to the regulation of phosphorylation.Under normal physiological conditions, the phosphorylation and the dephosphorylation were in a state of dynamic balance. At present,tau protein can be phosphorylated by more than 20 kinds of kinases. Among those, guided by proline glycogen synthesis kinase 3 (glycogen synthase kinase, GSK-3) and cell cycle dependent protein kinase 5 (CDK5) were more important. Phosphorylated tau protein could lead to early symptoms of neurodegenerative diseases, such as:synaptic loss,neuronal axons transport defect, neural inflammation and so on. Phosphorylated tau protein further leaded to PHFs, which was the main pathological characteristic of AD and other tau protein disease. So it has important practical significance that targeting the tau protein to explore treatment of neurodegenerative disease including the PD.1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a kind of white powder nerve agents, with a specific damaged effect on substantia nigra dopamine neurons, could copy the clinical manifestations and pathological features of PD, such as:paralysis symptom, apoptosis of dopaminergic neurons in substantia nigra, the depletion of striatal dopamine, behavioral damage, and could cause tau protein hyperphosphorylation. MPTP,as a ideal agent,was used to study the PD. Mechanism of MPTP damaging nerve was that MPTP was transformed into 1-methyl-4-phenyl-pyridiniumion (MPP+) that was the active product by MAO-B in glial cells.A patent in the United States (USA, US7629374B2, use of aminoindazole derivatives for the inhibition of tau phosphorylation) has shown that amino indazole derivatives could inhibit the tau phosphorylation, but did not illustrate which specific compounds with inhibition of tau phosphorylation. Basing on the principle that similar structure compounds might have a similar effect, we screened a series of indazole derivatives that might inhibit tau phosphorylation. We found 6-amino-1H-indazole fortunately. Then,whether 6-amino-1H-indazole has protective effect on PD dopamine neurons in vivo and vitro or not? If so, how to protect? Whether the molecular mechanism is related to inhibit the tau protein phosphorylation? Based on these problems, this topic performed a series of studies.MethodsWe used SH-SY5Y cells damaged by MPP+to build the PD cells model in vitro, and C57BL mice were used to establish PD animals model in vivo by intraperitoneal injection of MPTP. Based on these two PD models,we performed our studyIn vitro experiment:We used MTT analysis to detect the effect of 6-amino-1H-indazole pretreatment on decreased cell viability induced by MPP+in SH-SY5Y cells; Tyrosine hydroxylase (TH) immunocytochemistry staining was used to detect the effect of 6-amino-1H-indazole pretreatment on apoptotic dopaminergic neurons induced by MPP+; Hoechst33258 nuclear staining was used to observe the effect of 6-amino-1H-indazole pretreatment on apoptotic nuclear morphology induced by MPP+.In vivo experiment:we tested the effect of 6-amino-1H-indazole pretreatment on incubation and duration period of PD symptoms by observing the PD symptoms; The tyrosine hydroxylase (TH) immunehistochemical staining was used to detect the protection of 6-amino-1H-indazole on midbrain dopaminergic neurons in PD mice; HPLC was used to detect the effect of 6-amino-lH-indazole pretreatment on striatal dopamine levels in PD mice; Behavioral experiment was used to test improvement of behavior damage of the PD mice by 6-amino-1H-indazole pretreatment.Mechanism research:western blot and immunohistochemistry were used to detect mechanism of dopamine neural protection of 6-amino-lH-indazole pretreatment in PD models.ResultsAn apoptotic model was successfully established in SH-SY5Ycells induced by MPP+ in vitro, and we explored the protection of 6-amino-lH-indazole on vitro dopaminergic neurons. To explore the best concentration of MPP+,we first used the 0-1500 μM MPP+ on SH-SY5Y cells for 24-72h. Experiment results showed that cell activity of SH-SY5Y cells decreased as increased concentration of MPP+. At 1000 μM MPP+ for 72h, cell activity decreased to 50%. As increase of concentration, cell survival rate no longer declined obviously. Therefore, we selected 1000 μM MPP+ for 72h on SH-SY5Y cells as a condition of establishing cell model.0-200 μM different concentrations of 6-amino-1H-indazole were solely treated the cells. We found the effect of 0-100 μM 6-amino-lH-indazole on the change of the cell survival rate had no statistical significance,while 200 μM 6-amino-1H-indazole decreased obviously the survival rate.6-Amino-lH-indazole did not promote or inhibit the growth of cells. Before 1000 μM MPP+treatment,0-200 μM 6-amino-lH-indazole pretreated SH-SY5Y cells for 2h. Comparaed to the MPP+ group, cell survival rate in medication group increased significantly, and the cell survival rate was about 96% of the control group at 100 μM. TH immunochemical staining was used to detect the number of residual dopaminergic neurons. The results shown that MPP+induced a lot of loss of dopamine neurons and decreased cell volume. However,the numbers of TH positive cells were significantly increased and increase of cell volume after 100 μM 6-amino-1H -indazole pretreatment. We performed Hoechst 33258 nuclear staning to further validate that 6-amino-1H-indazole had anti-apoptosis effect.The nuclear in control group are distributed in uniform dispersion. However,nuclear in model group were fracture, gathered apoptosis morphology.6-Amino-lH-indazole could significantly inhibited the state of the nucleus apoptosis induced by MPP+.In our vivo experiment,we successfully prepared the PD animal model induced by MPTP in C57BL mice, and observed the effect of 6-amino-1H-indazole on apoptotic dopaminergic neurons and improved the damaged behavior in the PD mice. After MPTP injection,the incubation and duration period of PD symptoms were recorded. The MPTP group had the obviously PD symptoms,shorter incubation period (1.62±0.11 min) and longer duration period (21.86±0.21 min),compared to control group.6-Amino-1H-indazole pretreatment could significantly extend the incubation period of PD mice (5 mg/kg 6-amino-1H-indazole group:4.55±0.14 min; 7 mg/kg 6-amino-1H-indazole group:3.82±0.20 min), shorten the duration period (5 mg/kg 6-amino-1H-indazole group:13.18±0.27 min; 7 mg/kg 6-amino-1H-indazole group:18.00±0.34 min), and alleviated symptoms of parkinsonism. The substantia nigra compacta of midbrain was performed immunohistochemistry.We found that the number of dopamine neurons decreased significantly induced by MPTP. However,6-amino-1H-indazole pretreatment increased the numbers of dopamine neurons significantly. HPLC method was used to analyze the levels of striatal dopamine. We found that when compared with MPTP group, medication group significantly increased the levels of striatal dopamine. Traction and pole test were performed to detect behavior change of PD mice. In the pole test, compared with bradykinesia in MPTP model group, mice in 6-amino-lH-indazole group required less time to perform specified action and get higher scores. In traction experiments, the mice in control group performed freely and could grisp the wire with four hinds.The MPTP group with the weak hind legs could only use two forelimbs to seize the wire, or drop. The mice in 6-amino-lH-indazole group could use one or two hind legs to seize the wires and scored higher. In our experiment, we found that compared to the high dose group(7 mg/kg 6-amino-1H-indazole), low dose group (5 mg/kg 6-amino-lH-indazole) presented the better protective action on dopamine neurons and resistance to behavioural damage.We used western blot and immunohistochemistry to detect possible mechanism of dopaminergic neural protection of 6-amino-lH-indazole in PD models. In cell western blot analysis, we found that MPP+induced the tau phosphorylation at the peak at 8h. However,100 μM 6-amino-1H-indazole could obviously inhibit the MPP+-induced tau phosphorylation. In the fluorescence double staining,TH expression in control group was obvious and phosphorylated tau protein expression was weak. However, in MPTP group, expression of phosphorylated tau protein in dopaminergic neurons significantly increased, compared with the control group.The numbers of neurons reduced. While expression of phosphorylated tau protein was reduced in the lower dose drug groups.Experimental results showed that 6-amino-1H-indazole played a role of neuroprotection might be related to inhibition of tau phosphorylation.Conclusions1.6-Amino-lH-indazole had protective effect on apoptosis of SH-SY5Y cells induced by MPP+2.6-Amino-lH-indazole had protective effect on dopaminergic neurons in the substantia nigra in MPTP PD mice. It increased the striatal dopamine levels and protected the damaged behavior3.MPP+/MPTP induced the phosphorylation of tau in vivo and vitro. 6-Amino-lH-indazole inhibited the tau phosphorylation and protected dopaminergic neuronsTo sum up, this study integratly used molecular biology, morphology, biochemistry, behavioral science and other research methods to confirm that protection of 6-amino-1H-indazole on the dopamine neurons in Parkinson’s disease, and explore the molecular mechanism might be related to inhibition of tau phosphorylation. The study laid a foundation for further studying of 6-amino-lH-indazole on the other nervous system diseases and the clinical application in the further... |
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