| Objective:This study was designed to evaluate the therapeutic effect of traditional Chinese medicine Congrong Jing Formulation (CRJ) on Parkinson’s disease. To investigate the molecular mechanism of CRJ to observe its effect on the JAK2/STAT3Pathway,and the expression of key regulatory factors in the JAK2/STAT3Pathway, such as JAK2,p-JAK2, STAT3,p-STAT3, Bcl-2, Bax. The aim of this study was to provide a theoretical basis for the clinical application of Congrong Jing Formulation.Method:Take MES23.5nerve cells at logarithmic phase for experiment.Cells were cultured in96-well plates at a concentration of5×104for24h, then intervened with different concentrations of MPP+for24h and48h, respectively. It was selected100umol/L for24hours to establish cellular PD model by MTT assay. Cells were cultured in96-well plates at a concentration of5×104.When the cells was established PD model, different concentrations of CRJ aqueous extract was treated for24h and48h, respectively. Concentrations of100,200,250ug/ml were seted low, medium and high dose group intervened for24hours for subsequent experiments. The MES23.5dopaminergic neurons were randomly divided into normal group, model group, CRJ low, medium and high dose groups.In addition to the normal group, model group and each other treated groups were given a final concentration of100μmol/L of MPP+for24hours to build a model of Parkinson’s disease. The normal group and model group replaced with fresh medium, CRJ low, medium and high dose groups replaced with a final concentration of100,200and250ug/ml CRJ aqueous extract medium for24h. Cellular morphology of every group was observated by inverted phase contrast microscope. The expression of tyrosine hydroxylase was measured by immunocytochemistry staining; apoptosis rate in each group was tested by flow cytometry. The protein expression of JAK2, p-JAK2, STAT3, p-STAT3, Bcl-2, Bax was determined by Western blot.Results:MTT results showed that MPP+can significantly reduce the survival of dopaminergic neurons, and showed dose-and time-dependent phenomena. Within0-500mg/ml CRJ aqueous extract did not cause any cytotoxic effect on the cell viability. CRJ aqueous extract can significantly reduce toxicity induced by MPP+in dopaminergic neuronal cells and increase cell survival. Immunocytochemistry showed that the model group were significantly decrease the expression of tyrosine hydroxylase, there was a significant difference (P＜0.05) compared with the normal group. The expression of tyrosine hydroxylase at each dose of CRJ aqueous extract was significantly increased, especially in the high-dose group, there was a significant difference (P＜0.05) compared with the model group. Flow cytometry showed that the model group significantly increased apoptosis rate, there was a significant difference (P＜0.05) compared with the normal group. CRJ aqueous extract decreased the apoptosis rate in each dose group, there was a significant difference (P＜0.05) compared with the model group. Western blot showed that there was no difference (P＞0.05) in expression of p-JAK2, p-STAT3between the normal group and model group.Compare with the normal group and model group, the expression of p-JAK2and p-STAT3were significantly increased in each dose group of CRJ aqueous extract, especially in the high-dose group (P＜0.05).Conclusion:1. CRJ aqueous extract has neuroprotective effects.It can reduce MPP+-induced apoptosis and showed dose-effect relationship.2. CRJ aqueous extract can significantly improve tyrosine hydroxylase which is the key rate-limiting enzyme in the synthesis of DA, and showing dose-effect relationship.3. CRJ aqueous extract of have significant anti-apoptotic effects, can significantly increase JAK2, STAT3phosphorylation levels, can enhance the expression of Bcl-2protein, decrease the expression of Bax protein and showed dose-relationships. Therefore, the regulation of JAK2/STAT3signaling pathway may be one of the mechanisms of treatment of PD. |
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