| α-synuclein(α-syn) are the key pathological substances in Parkinson’s disease. The transmission of α-syn between nerve cells is one of the most important pathological mechanisms of the progression of this disease. And the secretion of α-syn plays a central role in α-syn transmission. However, there are seldom pharmacological researches on blocking this pathology so far. Baicalein(Baicalein, BAI), an active ingredient of a traditional Chinese medicine called Scutellaria baicalensis Georgi, can bind to α-syn, prohibit its abnormal aggregation, degrade its aggregates, and alleviate its cytotoxicity, but reports of this drug on blocking α-syn secretion are pretty scarce until the recent. We predicted that BAI is possible to block the secretion of α-syn. Here, we established the stable SN4741 cells overexpressing α-syn by lentivirus infection as the experimental models to explore the effects of BAI on blocking α-syn secretion. Objective:1. To establish the stable SN4741 cells overexpressing wild type(WT) or A53 T mutant type α-syn.2. To explore the effects of BAI on blocking α-syn secretion. Methods:1. First, full-length coding sequence of homo SNCA-WT was amplified from cDN A by PCR, and then the fragments were inserted into p IRES2-EGFP vector, which was used as the intermediate vector to construct the SNCA mutants at A53 T site. Secon d, the SNCA-WT and SNCA-A53 T fragments were inserted into p LVX-IRES-PURO vec tor to construct two lentivirus expression vectors which were p LVX-SNCA-WT-IRES-P URO vector and p LVX-SNCA-A53T-IRES-PURO vector, respectively. At the same time,p LVX-EGFP-IRES-PURO vector was also constructed as control. Third, the three vec tors were cotransfected with the viral packing plasmids into 293 T cells, respectively. F ourth, normal SN4741 cells were infected by lentiviruses carrying SNCA-WT, SNCA-A53 T or EGFP, respectively Finally, cells infected by lentiviruses carrying different gene s were administrated by Puromycin in order to select stable cells overexpressing WT α-syn., A53 T α-syn. or EGFP.2. To administrate normal SN4741 cells with different concentrations of BAI, and then the cell viability was detected by MTT to explore the appropriate dose of BAI used in the following experiments.3. To treat the normal SN4741 cells and the stable SN4741 cells overexpressing EGFP, WT or A53 T α-syn with BAI, which were the NORM+BAI group, the EGFP+BAI group, the WT+BAI group and the A53T+BAI group, and the control groups of the same genotypes were established respectively, which were the NORM group, the EGFP group, the WT group and the A53 T group, at the same time. Western blot was used to detect the level of α-syn in culture media(CM) and in cell lysates(CL), the level of Flotillin-1 and Lamp-1, which are the makers related to α-syn secretion, and the level of LC3 B and LAMP2, which are the makers related to α-syn degradation, after BAI administration for 6h, 12 h and 24 h respectively in order to explore the effects and mechanisms of BAI on blocking α-syn secretion. Results:1. The results of DNA sequencing indicated that all the cloning coding squences of SNCA-WT, SNCA-A53 T and EGFP were the same as the corresponding template sequences. The results of qPCR showed that the relative expression level of SNCA of SN4741 cells infected by lentiviruses carrying SNCA-WT or SNCA-A53 T gene increased significantly. The SN4741 cells infected by lentiviruses carrying EGFP gene could express EGFP, which was confirmed by fluorescence microscope.2. The results of MTT demonstrated that as compared with the NORM group, the cell viability of all the BAI groups rose significantly after BAI administration for 6h(P<0.01) except for 1μM group and 3μM group; the cell viability of all the BAI group went up sharply after BAI admistration for 12h(P<0.01); but significantly increased cell viability was only observed in the 200 uM group and 400 uM group after BAI administration for 24 h(P<0.01).3. In comparison with the control groups of the same genotypes, the level of α-syn in the CM of the WT+BAI group and the A53T+BAI group reduced significantly after BAI administration for 6h or more, whereas the level of α-syn in the CL of these groups rose after BAI administration for 6h and 12 h, and declined after BAI administration for 24 h, of which significant differences were observed in the level of α-syn of all the groups administrated by BAI at different time points except for those in the WT+BAI 12 h subgroup, which almost had significant difference(P≈0.063).In comparison with the control groups of the same genotypes, the level of Flotillin-1 in the CL of SN4741 cells of each genotye all decreased after BAI administration for 24 h, of which the NORM+BAI group and the WT+BAI group were significantly different from their control groups of the same genotypes, and the EGFP+BAI group was near statistical different from its control group(P≈0.060), while the A53T+BAI group had no significant difference from its control one. At the same time, the level of LAMP1 in the CL of the NORM+BAI group, the EGFP+BAI group and the WT+BAI group all descended, but no significant difference was found in all those decrements.In comparison with the control groups of the same genotypes, the concentration of LC3 B in the CL of the A53T+BAI group significantly declined after BAI administration for 6h. The level of LC3 B in both the NORM+BAI group and the A53T+BAI group significantly decreased after BAI administration for 12 h. The reduction of LC3 B level in all the BAI administrated groups was observed after BAI administration for 24 h, of which all the BAI administrated groups differed significantly from their control ones except for the WT+BAI group.In comparison with the control groups of the same genotype, the concentration of LAMP2 in the CL of the NORM+BAI group and the EGFP+BAI group both increased, whereas that of the WT+BAI group and the A53T+BAI group both decreased, of which the NORM+BAI group and the A53T+BAI group had significant differences. The level of LAMP2 in the CL of all the BAI administrated groups had a reduction after BAI administration for 12 h and 24 h when compared with their control groups of the same genotypes, of which the EGFP+BAI 12 h subgroup, the WT+BAI 24 h subgroup and the A53T+BAI 12 h subgroup and its 24 h subgroup differed significantly from their control groups, respectively. Conclusion:1. Stable SN4741 cell lines overexpressing EGFP, WT or A53 T α-syn are established successfully.2. The concentration of 100μM BAI is determined to be used in the following experiments.3. BAI can reduce the level of α-syn in CM of SN4741 cells overexpressing WT/A53 T α-syn, enables the level of α-syn in CL of these cells elevate in the early stage and decline in the late stage of BAI administration, and reduces the level of intracellular Flotillin-1, LC3 B and LAMP2, but has no significant effect on LAMP1, which are related to α-syn secretion and degradation, respectively. These results suggest that BAI is possible to block α-syn secretion. |
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