The Therapic Effect And Its Mechanism Of Chinese Medicine "Chanfuning" On Parkinson's Disease

Parkinson's disease (PD) is a widespread neurodegenerative disorder, while its etiology is still unknown. Besides the L-dopamine (L-DA), the current strategy is the neuroprotective therapy. Some experiments reported that the Chinese medicine had obvious therapeutic effect on PD, which is regarded as one of the neuroprotective therapy. However, there is few study on the mechanism of Chinese medicine's therapeutic effect on PD on the cellular and molecular level.Chinese medicine "Chanfuning" has a beneficial effect on treating PD, which has proved by the twenty-year's clinical application. It could not only improve the clinical symptoms of PD patients, but also remarkably enhance the immune system function. Chanfuning combined with the L-DA has much better effects than the L-DA, which could reduce the ratio of side effect.In order to study the mechanism of Chanfuning on treatment of PD, we used the hemilateral rat model of PD in vivo and dopaminergic(DA) neurons in vitro injuried by 6-OHDA to imitate the progression of PD. Immunohistochemical staining, RT-PCR, serum pharmacologic method, laser scanning confocal microscopy(LSCM), and so on were performed in order to probe into the effect of Chanfuning on DA neurons, and identify its antioxidation and anti-apoptosis mechanism. Our study has gotten some experimental evidences on the effect of Chanfuning on treating PD, and could provide new therapy idea for PD patients. All the experiments were divided into 5 parts as follows:Objective: To study the therapeuti effect of Chinese medicine Chanfuning on rat model of Parkinson's disease (PD).Methods: After injecting 6-OHDA into the right striatum of rat for 2 weeks, we obtained the hemi-lateral model of PD, All rats were induced the rotational behavior after injecting apomorphine subcutaneously. The control group was set up by injecting isodose physiological saline as pseudo-operation rats. All the animals were divided into 3 groups: control group, model group, and Chanfuning-treated group. Each group has 6 rats. The control and model groups were treated with physiological saline for 30 days, and the rats in Chanfuning-treated group were treated with Chinese medicine Chanfuning decoction for 30 days. The rotational behavior was evaluated by recording the numer of apomorphine-induced turns. We used tyrosine hydroxylase (TH) immunohistochemical staining to investigate the numbers of dopaminergic neurons in substantia nigra compacta(SNc) and the calibrated optic density (COD) value of TH positive fiber in stritum. The data were analyzed using SPSS 10.0 statistics software, results were typically presented as means ±SEM. Statistical significance was assessed using a one-way ANOVA, and significance was set at P<0.05.Results:1. Chanfuning can ameliorate the rotational behavior of Parkinsonian rats, comparing with the control group, P<0.01.2. TH positive neurons in right SNc and TH positive fibers in right striatum of the PD model group significantly decrease (P<0.01). After being treated with Chanfuning, they can partly be restored.Conclusions: Chanfuning had partial therapeutic effect on model rats of PD.Part 2: The effect of Chanfuning on contents of GSH and MDA in SNc of rat model of PDObjective: To illustrate the anti-oxidative effect of Chanfuning on rat model ofPD.Methods: The experimental rats were divided into 3 groups according to part 1. In deeply anesthetized condition, SNc was taken out, made 10%homogenate, then MDA and GSH contents in it was meastured by spectrophotometer in order to observe the effect of Chanfuning on oxidative stress reaction.Results: In SNc of model group, the content of MDA showed dominant increase and the GSH decrease (P<0.01). While in Chanfuning-treated group, the results is totally reverse: MDA decreased and GSH increased (P<0.01).Conclusions: 6-OHDA-induced oxidative stress results in the depletion of intracellular GSH, while Chanfuning can maintain GSH content and play an important role in antioxidation. So, Chanfuning can protect neurons from injury of free radical.Part 3: The effect of Chanfuning on calbindin D28K mRNA in SNcof rat model of PDObjective: To investigate the change of calbindin D28K mRNA in SNc of rat model of PD after treated by Chinese medicine Chanfuning.Methods: The experimental rats were divided into 3 groups as part 1 described. In deeply anesthetized condition, the substantia nigra of midbrain of rats were immediately taken out. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was employed to normalize the expression of calbindin D28K mRNA in those SNc tissues. Statistical analysis was the same as part 1.Results: In model group SNc, calbindin D28K mRNA significantly decreased (P<0.01). While in Chanfuning-treated group, calbindin D28K mRNA significantlyincreased (PO.05).Conclusions: Chanfuning can enhance the content of calbindin D28K mRNA and inhabit Ca2+ influx to prevent the DA neurons from apoptosis.Part 4: Protective effect of the serum with Chanfuning decoction on mesencephalic dopaminergic neurons in vitro and its mechanismObjective: To observe the influence of the serum containing Chanfuning decoction on cell growth and intracellular Ca2+ of mesencephalic dopaminergic neurons in vitro.Methods: The Chinese medicine Chanfuning serum was made by serum pharmacological method. Pregnant Wistar rats were anesthetized with an overdose of pentobarbital, then embryonic day 15-16 (E15-16) embryos was obtained, from which the ventral mesencephalon (VM) was taken out into cold D-hanks buffer. The VM cells were trypsinized to single ones and maintained in DMEM containing 10% fetal calf serum. The VM cells were cultured in 24-well and 96-well plates respectively, and kept in a water-jacketed tissue culture incubator at 37°C with 5%CO2/95% air. To set up the PD model in vitro, 100MM 6-OHDA were added into the medium of dopaminergic neurons for 24 hours. There were 3 groups: (1) control group was treated with control rat serum; (2) 6-OHDA-treated group was treated with control rat serum and 100MM6-OHDA; (3) Chanfuning-treated group was treated with drug-treated rat serum and 100MM6-OHDA. MTT assay method was used to determine the cell activity. The tyrosine hydroxylase (TH) immunocytochemistry staining was applied to investigate the number, the length of the longest neurite, and soma area of dopaminergic neurons. Changes in intracellular calcium concentration were examined by laser scanning confocal microscopy(LSCM). Biochemical assays were used to measure the levels of intracellular GSH and MDA contents.Results:1. Chanfuning rat serum significantly attenuated 6-OHDA-induced neurotoxicity in primary cultured mesencephalic cells with a dose-dependent manner. Cell viability in Chanfuning serum treated group increased significantly (PO.01).2. After being damaged by 6-OHDA, the number of surviving TH- positive cells in Chanfuning serum treated group increased significantly; the mean longest neurite length of TH-positive neurons exposed to the medium with Chanfuning serum was much longer than in the control serum cultures. There was no significant difference in the cell soma area.3. The concentration of intracellar Ca2+ in cells treated by Chanfuning serum was significantly higher than treated by control serum.4. Contrasting with the depletion of GSH induced by 6-OHDA, Chanfuning serum preserved the intracellular GSH content. Further research revealed that Chanfuning serum reduced the level of MDA dramatically and protected neurons from oxidative damage.Conclusions: Chanfuning serum may have direct nutrition effects on dopaminergic neurons and protect them from injuries induced by 6-OHDA. By inhibition of calcium influx, Chanfuning serum could restrain the apoptosis of DA neurons.Part 5: Antiapoptosis effects of Chanfuning serum on PC12 cellsinjuried by 6-OHDAObjective: To study the inhibitory effects of Chanfuning serum on the apoptosis of PC 12 cells induced by 6-OHDA.Methods: The Chinese medicine Chanfuning serum was made by serum pharmacological method. 6-OHDA and Chanfuning serum were added into the medium of PC 12 respectively, and then MTT method was used to assay the cellviability, meanwhile tyrosine hydroxylase (TH) and Caspase-3 immunocytochemistry staining conducted to measure the content of TH and count the number of apoptosis cells.Results: Comparing with 6-OHDA treated group, the PC 12 cell viability and the calibrated optic density (COD) of TH positive cells increased significantly in Chanfuning serum treated group (PO.01), and the number of Caspase-3 positive cells decreased (PO.01).Conclusions: Chanfuning serum may have direct nutritious effects on PC 12 cells, protecting them from the injury induced by 6-OHDA and producing anti-apoptosis effect to some extent. Conclusions and significance:Our research found that Chinese medicine "Chanfuning" could protect the 6-OHDA-induced dopaminergic neurons from apoptosis in rat model of PD and the serum containing Chanfuning decoction had direct nutritional and protective effects on dopaminergic neurons and PC 12 in vitro. Its mechanism could be elucidated from two aspects: it could increase the content of GSH and degrade oxygenation resulting from oxygen free radical. On the other hand, Chanfuning could enhance the calbindin D28K mRNA content and inhibit the calcium influx to prevent the DA neurons from apoptosis.From in vivo and in vitro research, we systematically studied the therapic effect on PD of Chinese medicine "Chanfuning" which has the definite clinical curative effect. On cellular and molecular level, our study provided Chanfuning with some experimental evidence on treating PD and would shed light on the therapic mechanism of PD patients as well.